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Article

Identification of Proteins Associated with Stably Integrated Maize b1 Tandem Repeat Transgene Chromatin

by
Jason S. Lynn
†,
Kathryn M. Koirtyohann
,
Yacob B. Gebreab
,
Jaliyah Edwards
and
Karen M. McGinnis
*
Department of Biological Science, Florida State University, Tallahassee, FL 32304, USA
*
Author to whom correspondence should be addressed.
†
Current Address: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
Plants 2025, 14(12), 1863; https://doi.org/10.3390/plants14121863
Submission received: 11 April 2025 / Revised: 6 June 2025 / Accepted: 14 June 2025 / Published: 17 June 2025
(This article belongs to the Section Plant Molecular Biology)
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Abstract

:
The control of gene expression by cis-regulatory DNA sequences is a conserved genomic feature. The maize booster1 gene (b1) is a naturally occurring locus that serves as a mechanistic model for the control of gene expression from a distal cis element and a form of allelic interactions called paramutation. Two epi-alleles of b1 produce distinct pigmentation phenotypes correlated with transcriptional enhancement and the silencing of b1. These transcriptional dynamics depend on a hepta-tandem repeat sequence located 100 kb upstream of the b1 locus. In the heterozygous condition, the B′ epi-allele paramutates B-I, heritably converting the B-I epi-allele to the epigenetic state and expression level of B′, producing lightly pigmented plants. To identify b1TR-associated proteins, we used a targeted chromatin immunoprecipitation approach with a stably integrated transgenic b1TR locus. Applying a conservative filtering strategy, we detected several expected factors, including RNA Polymerase II, as well as the novel putative DNA-binding proteins ZAG4 and DDT4. ZAG4 and DDT4 activated GAL expression using b1TR as bait in yeast one-hybrid, supporting their potential interaction with this sequence. The identification of proteins uniquely associated with the UAS::b1TR chromatin provides insight into potential b1 regulatory factors and offers a foundation for future studies to investigate their roles in gene regulation.

1. Introduction

Transcription is spatiotemporally controlled, requiring the contribution of DNA regulatory sequences and chromatin-interacting proteins. Historically, cis-regulatory elements have been studied via genetic approaches, such as sequence deletions to test for necessity, and by the insertion of those sequences at heterologous genomic or reporter gene locations to test for sufficiency (reviewed in [1]). The investigation of DNA–protein interactions has been performed using in vitro or heterologous assays, such as the yeast one-hybrid assay or DNA-affinity purification [2,3], which can identify proteins that bind to cis-elements and determine the minimal sequences required for interaction. However, none of these methods maintain the in vivo context of chromatin and are therefore not necessarily representative of the relevant biochemical context of the regulatory sequences.
Chromatin-immunoprecipitation DNA-sequencing (ChIP-seq) and related methods are widely used to map the binding sites of a wide range of chromatin-interacting proteins, such as transcription factors and histone modifications. These approaches query the DNA associated with specific proteins and do not allow for the de novo identification of unknown proteins interacting with specific cis elements. To better understand the role of specific proteins at a particular cis-regulatory element, locus-specific chromatin capture-based proteomic methods are required. A few methods have been published seeking to identify proteins associated with well-characterized genetic elements in model organisms and cell lines [4,5,6,7]. More recent attempts to identify single-locus proteomes using modified CRISPR/Cas9 methods have allowed the characterization of protein factors at some chromosomal sequences [8,9,10,11,12]. Most of these approaches have been focused on characterizing proteins associated with large, highly repetitive centromere and telomere regions rather than low copy or unique loci.
The maize b1 hepta tandem repeat (b1TR) is a tissue-specific cis-regulatory element located on the short arm of chromosome 2 that can exist in distinct states associated with two genetically identical, but epigenetically distinct, alleles of b1 referred to as B-Intense (B-I) and B-Prime (B′). In the B′ configuration, the hepta-repeat b1TR acts as a silencer, leading to low B1 expression and lightly-pigmented sheath and husk tissue; in the B-I configuration, the hepta-repeat b1TR is in a distinct epigenetic state and acts as an enhancer leading to high B1 expression and deeply-pigmented sheath and husk tissue [13,14,15,16]. This phenomenon is subject to genetic variation, with copy number variation of naturally occurring and transgenic versions of the b1TR sequence playing a key role in epigenetic regulation and inheritance. Alleles of b1 with less than three repeats at b1TR do not display enhancer or silencer activity while five or more repeats at b1TR are required for transcriptional enhancement and the paramutation of b1 (reviewed in [17]). The expression of the b1 gene is correlated with changes in the epigenetic state of b1TR [16] and differential tissue-specific long-range intra-chromosomal contacts [15]. Although the establishment of paramutation is expected to occur during meiosis (Coe 1959 [18]), the transmission of this epigenetic state must be maintained throughout development because the B′ allele remains transcriptionally silent in the sheath and husk tissues of the mature plant [19]. Genetic analysis shows that proteins predicted to be components of the RNA-directed DNA-methylation pathway (RdDM) are differentially required for the establishment and maintenance stages of paramutation, meaning that different individual complexes and proteins might bind to and act on b1TR at different stages of paramutation [20,21,22,23,24,25,26,27,28].
The identification of b1TR-interacting proteins will therefore be important in understanding the mechanisms underlying these distinct regulatory processes. Transgenic versions of b1TR are sufficient for the silencing and paramutation of the endogenous b1TR enhancer from non-allelic positions [29], suggesting that b1TR-interacting regulatory proteins bind transgenic versions of this sequence. To identify proteins that interact with b1TR, we used a transgene-mediated chromatin-immunoprecipitation mass-spectrometry-based proteomics (ChIP-MS) approach that utilizes the yeast-derived GAL4 DNA binding protein’s specificity for its binding site and the Upstream Activation Sequence (GAL4-UAS) [30] to enrich for b1TR-sequence-associated chromatin-interacting proteins. We show that a transgenic copy of b1TR cloned adjacent to GAL4-UAS (UAS::b1TR transgene) is sufficient to induce silencing of B-I in stably transformed plants. Using this ChIP-MS approach, we detected peptides of components of the Mediator complex and the RNA Polymerase II transcriptional complex and generated a list of RNA- and DNA-binding proteins that uniquely associate with the UAS::b1TR chromatin. Two of these putative DNA binding proteins, DDT4 and ZAG4, were able to activate transcription of the GAL reporter locus using b1TR as bait in a yeast one-hybrid assay.

2. Methods

2.1. Maize Plasmid Construction

The UAS::b1TR, UAS::empty, and FLAG-GAL4 constructs were generously gifted by Vicki Chandler, Jan Brzeski, and Kasia Brzeska. The UAS::b1TR construct was generated by fusing the b1TR sequence (7 × 853 bp tandem repeat) from the pBΔ construct [29] to an array of ten yeast minimal GAL4-UPSTREAM ACTIVATION SEQUENCE (UAS; 10 × 90 bp UAS) corresponding to the NCBI accession CP036475.1 (Saccharomyces cerevisiae strain ySR128; Chr13:599010-599024). The UAS::empty construct was generated by excising the 6684 bp hepta-b1TR sequence from the UAS::b1TR construct by using restriction digest and re-ligation. The FLAG-GAL4 construct encodes an N-terminal-FLAG:GAL4 DNA binding domain corresponding to the NCBI accession CP033485.1 (S. cerevisiae strain y169; Chr16:81014-81471) and the Alcohol dehydrogenase 1 (adh1) intron. Expression is driven by the maize ub1 promoter and is terminated by the OCS 3′ signal sequence. Additionally, all constructs contain glufosinate resistance under the control of the minimal Cauliflower mosaic virus 35S promoter (CaMV 35S) and the Tobacco mosaic virus Omega leader and are terminated by the nopaline synthase polyadenylation signal (NOS).

2.2. Transformation of Maize Stocks

The transformation of maize (Zea mays) plants was performed by the Iowa State University Plant Transformation Facility using biolistic bombardment of Hi-II embryos [31]. Glufosinate resistant calli were screened for transgene insertion by PCR. Plantlets were regenerated and backcrossed with Hi-II to generate plants with single transgene insertions. Transgenic Hi-II plants were crossed to B-I/B-Peru plants to generate segregating progeny for the transgene and B-I. We refer to the B allele of Hi-II as B-neutral (b-N) because it does not confer intense pigmentation to the sheath and husk tissues nor does it participate in paramutation.

2.3. Pigmentation Analysis of the UAS::b1TR and UAS::Empty Transgenes for Silencing of B-I

The silencing abilities of UAS::b1TR constructs were determined by pigmentation scoring of sheath and husk tissues in a blind, randomized field experiment. A total of 179 transgenic plants containing UAS::b1TR and 203 non-transgenic plants were segregating for B-I/b-N or B-Peru/b-N. 167 transgenic plants containing UAS::empty, and 158 non-transgenic plants were segregating for B-I/b-N or B-Peru/b-N. B-I and B′ plants were used as controls for herbicide scoring and pigmentation. In addition to b1, a functional copy of Pl1 and the full complement of anthocyanin biosynthetic genes were required for complete pigmentation. All plants contained at least one Pl1 allele, conferring strong pigmentation as B-I/B-Peru parents were homozygous for Pl1. Each individual plant was numbered and tagged, and the genotypic identity of the families was hidden from observers. At maturity (R1, silks exposed), the plants were scored for sheath and husk color by three independent blinded observers using a color scale of 1–5 with 5 being darkest. Plants with a color score <4 were considered light, while plants with a color score of ≥4 were considered pigmented. Finale® herbicide (1.6% v/v in water; BASF) was applied to a leaf from every individual to differentiate transgenic and non-transgenic plants by scoring for the presence or absence of leaf tissue damage from herbicide application. Any observations that produced a standard deviation ≥1 for either sheath- or husk-color score across three independent observers were considered erroneous and were removed from subsequent analysis.

2.4. Sample Growth Conditions and Sample Collection

Maize plants were grown in a greenhouse with supplemental lighting to extend the day length. Finale® herbicide (1.6% v/v in water; BASF) was applied to the leaves to screen for transgenic plants with a glufosinate resistance gene. Herbicide-resistant plants were genotyped using transgene-specific primers KM1684 (5′-GCGCTTTCTCATAGCTCAC-3′) and KM1687 (5′-AGTCGTGTCTTACCGGGTTG-3′). At maturity (R1, silks exposed), the plants were scored for pigmentation, and sheath tissue was dissected, snap frozen in liquid nitrogen, and stored at −80 °C.

2.5. FLAG-GAL4 Chromatin Immunoprecipitation and Mass-Spectrometry from Maize Sheath

For each biological replicate, 55–60 g of fresh sheath tissue was dissected from the mature plants and snap frozen in liquid nitrogen. Frozen tissue was then ground in liquid nitrogen by using a mortar and pestle. The tissue was further disrupted using a Waring Laboratory grade blender for 5 min on “low” in 300 mL of cross-linking nuclei isolation buffer 1 (10 mM Tris-HCl pH 8.0, 400 mM sucrose, 10 mM sodium-butyrate, 0.1 mM phenylmethylsuflonyl fluoride (PMSF), 5 mM β-mercaptoethanol, 0.33 µL/mL plant protease inhibitor cocktail (PIC, Sigma-Aldrich, St. Louis, MO, USA), 1% v/v formaldehyde). After homogenization and lysis, nuclear isolation was performed as previously described [32]. The chromatin was then fragmented with 60 units/mL of micrococcal nuclease (MNase; Worthington Biochemical, Lakewood, NJ, USA) to generate a distribution of nucleosomal bands that were mostly smaller than 1 kb but larger than 350 bp in length. Isolated soluble chromatin from two biological replicates (whole sheath tissue from individual plants) was used for each transgene combination, UAS::b1TR; FLAG-GAL4, UAS::empty; FLAG-GAL4, and—(no UAS transgene); FLAG-GAL4. Soluble chromatin from each biological replicate was diluted and incubated overnight at 4 °C with rotation with 4 µg of FLAG antibody (F3165, Sigma-Aldrich, St. Louis, MO, USA). An aliquot of soluble chromatin from the same biological replicate was incubated overnight at 4 °C with rotation with 20 µg of isotype control mouse IgG (I5381, Sigma-Aldrich, St. Louis, MO, USA) to identify common, non-specific proteins. Antibody–antigen complexes were captured using Dynabeads Protein G as per the manufacturer’s instructions (ThermoFisher, Bohemia, NY, USA). Beads were pooled and washed thrice with 750 µL of bead wash buffer 1 (0.01% v/v Tween-20, PIC in 1× PBS), thrice with bead wash buffer 2 (1× PBS, PIC), and twice with 100 mM ammonium bicarbonate before being transferred to a new 1.5 mL Protein Lo-Bind tube (Eppendorf, Hamburg, Germany) for a third wash. The supernatant was completely removed, and the beads were incubated in 20 µL of 10 ng/µL trypsin (ThermoFisher, Bohemia, NY, USA) in 100 mM ammonium bicarbonate. Digests were incubated for 4 h at 37 °C with occasional mixing by vortex. After digestion, the supernatant was collected off the magnetic beads and processed using C18-spin columns (ThermoFisher, Bohemia, NY, USA). The samples were lyophilized and the peptides were sequenced using the Thermo Q-Exactive LC-MS/MS (ThermoFisher, Bohemia, NY, USA) instrument as described in [33]. The aligned peptides were loaded into Scaffold ([34]; v4.10) for the visualization of identified proteins and statistical analysis. The candidate proteins selected for subsequent analysis had at least 1 peptide detected, a protein identification threshold set at 90.0%, and a peptide threshold at 95.0%. Randomized amino acid sequences of non-redundant proteins were used to determine the false discovery rate of the identified proteins across all of the samples (FDR = 1.2%).

2.6. ChIP-qPCR Enrichment Analysis of FLAG-GAL4 at UAS::b1TR

ChIP-qPCR was performed in triplicate using sheath tissue-derived soluble chromatin from UAS::b1TR;FLAG-GAL4 that was incubated with FLAG (F3165, Sigma-Aldrich, St. Louis, MO, USA) and mouse IgG (I5381, Sigma-Aldrich, St. Louis, MO, USA) antibodies. After chromatin immunoprecipitation, Dynabeads beads were pooled and washed as described above. The ChIP-DNA was eluted and used as template in qPCR as previously described [32]. To differentiate enrichment at UAS::b1TR from the ChIP-qPCR signal derived from the endogenous b1TR, primers were used to target a region specific to the UAS::b1TR transgene that was not present elsewhere in the maize genome: KM1684 (5′-GGCGCTTTCTCATAGCTCAC-3′) and KM1687 (5′-AGTCGTGTCTTACCGGGTTG-3′).

2.7. Cloning for Yeast One-Hybrid Experiment

LacZ bait plasmid constructs (pVC4241-1) were cloned during a previous study [35] by the insertion of a b1TR fragment (base pairs 362–853 from one repeat unit and 1–361 from the next repeat unit) into a pLacZi reporter plasmid (Takara Bioscience (formerly Clontech), San Jose, CA, USA). Negative control bait plasmids (4231 b1TR-) were prepared through digestion of the LacZ bait with SalI and XhoI to remove the b1TR fragment followed by re-ligation. The positive control prey construct (KKY006/b1TR_pos) contains CBBP from pVC442 (now cpp2, Zm00001d031008) in pEXP-AD502 (Invitrogen, Waltham, MA, USA). The ZAG4 experimental prey construct (YGY001) was assembled via Gibson assembly with the NEBuilder HiFi DNA Assembly Cloning Kit (Ipswich, MA, USA). The CBBP cDNA in pVC442 was replaced by Zag4 (Zm00001eb120710) cDNA using gene-specific primers “zag4 orf_fwd” (5′-aaaaagcaggcttgtcgaccATGATGACCGATCTGAGCTG-3′) and “zag4 orf_rev” (5′-ctgggtacgcgtgcggccgcTCATCCGAGTTGGAGGGTC-3′). The DDT4 prey plasmid (KKY004) was assembled from pVC442 by replacing CBBP cDNA with a DDT4 (Zm00001eb329910) cDNA sequence.

2.8. Yeast Transformation

Yeast (Saccharomyces cerevisiae) cells were transformed using the lithium acetate protocol [36]. An initial overnight culture in 5 mL of liquid YPD medium was grown at 30 °C on a wheel, followed by the inoculation of 15 mL of fresh YPD to an OD600 of between 0.2 and 0.3 and incubation at 30 °C with shaking at 200 rpm until OD600 reached 1. The yeast culture was harvested via centrifugation, washed with 1 mL ddH₂O, and resuspended in 1 mL 100 µM LiAc. The cells were pelleted at top speed for 15 s and the supernatant was removed. They were then resuspended in 250 µL 100 µM LiAc, vortexed, and aliquoted (50 µL per tube). The transformation mix was added, which consisted of 240 µL PEG (50% w/v), 69 uL ddH2O, 36 µL 1 M LiAc, 10 µL ssDNA, and 5 uL plasmid DNA. The mix was vortexed using a micromixer for 1 min, incubated at 30 °C for 30 min, and heat-shocked at 42 °C for 30 min. The cells were pelleted at top speed for 30 s, and the supernatant was removed and resuspended in 200 uL ddH₂O and was spread on plates for incubation at 30 °C for 3–4 days.

2.9. Quantitative X-Gal Assay for Measuring β-Galactosidase Activity

The quantitative X-gal assay for measuring β-galactosidase activity was performed following the method described by Trimborn et al. [37]. All of the transformed yeast cells were plated on SD Ura-/Tr- except for KKY002 (Supplemental Table S3), which was plated on SD Ura- media, and incubated at 30 °C for 48 h. After incubation, the yeast colonies were harvested and resuspended in Z-buffer, and OD600 was measured. A standardized yeast suspension (OD600 = 5.0) was prepared and subjected to three freeze–thaw cycles to permeabilize the cells. A total of 50 µL of the lysate was combined with a 330 uL X-gal master mix (agarose, PBS, β-mercaptoethanol, and X-gal) in a 96-well plate, and the reaction was monitored at room temperature until a blue color developed. Pictures of the reaction were taken at regular intervals, and image analysis was conducted using Fiji software with the ReadPlate 3.0 plugin. β-galactosidase activity was calculated based on the absorbance values.

2.10. Bioinformatic Analysis of Identified Proteins

PantherGO [38] was used to analyze gene-ontology terms associated with the identified proteins. Unique Uniprot identifiers of proteins based on their transgene association were used as input for PantherGO gene list enrichment analysis. Gene co-expression network analysis of Ddt4 (GRMZM2G128176) was performed using the maize gene co-expression network with default settings [39] (https://www.bio.fsu.edu/mcginnislab/mcn/main_page.php (accessed on 10 July 2019). Protein–protein interaction network analysis of ZAG4 and DDT4 was performed using STRING with default settings [40] (https://string-db.org/ (accessed on 26 August 2019)). Interaction data was imported into Cytoscape v3.6.1 to generate network interaction plots. Jalview was used to visualize the pair-wise alignment of DDT4 and BPTF, and SMART protein domain graphics were used for visual reference of the protein domains.

3. Results

3.1. UAS::b1TR Triggers Silencing of B-I While UAS::Empty Does Not

To determine the effects of UAS::b1TR in the transgene-mediated silencing of B-I, we analyzed families of B-I/b-N plants segregating UAS::b1TR or UAS::empty transgenes for sheath pigmentation phenotypes (Figure 1A,B). Neutral alleles of B (referred to here as “b-N”) are recessive to B-I (active) and B′ (silenced), include only a truncated sequence at the b1TR locus, and do not enhance B1 expression or participate in paramutation (Coe 1959; [18,41]). Based on the genotypes and phenotypes of the parents used for these genetic experiments, lightly pigmented plants are only expected when plants are homozygous for B′ or b-N, B’/b-N heterozygous, when plants are transgenic for a sequence that induces silencing of the endogenous B-I gene, when spontaneous silencing of b1TR occurs in the plant, or when there is a genetic interaction with the Hi-II background of the transgenic parent (Figure 1C). Consistent with previously published work demonstrating the silencing of B1 by a b1TR transgene (Belele et al. [29]), B-I plants transgenic for UAS::b1TR were more often lightly pigmented (89.9%; n = 179; p = 0.1785) than with UAS::empty (64.7%; n = 167; p = 4.98 × 10−6) or any non-transgenic siblings segregating within families as determined in a blind, randomized field pigmentation scoring experiment (Figure 1D). Although this result does not exactly match the expectation of silencing in every transgenic plant, statistical analysis supports the hypothesis that the transgene induces the silencing of B-I in the analyzed progeny (Table 1). This suggests that UAS::b1TR interacts with trans-silencing machinery while UAS::empty likely does not. Although slightly higher-than-expected rates of light plants (expect 50% light based on genetic segregation, Figure 1C) in UAS::empty and non-transgenic siblings were observed, chi square analysis suggests that there is a high likelihood that this may have been caused by extraneous factors which may include insufficient sample size, genetic contamination, errors in genotyping, unpredicted genetic interactions between Hi-II and B-I, and/or observer error in color scoring in the field (Table 1).

3.2. Identification of Proteins Purified by FLAG-GAL4 ChIP-MS from Transgenic Plants

To identify UAS::b1TR-interacting proteins, we performed anti-FLAG ChIP followed by mass-spectrometry using chromatin isolated from whole-plant sheath tissue from heterozygous B-I plants transgenic for FLAG-GAL4 and UAS::b1TR (B-I/b-N; UAS::b1TR/-; FLAG-GAL4/-), FLAG-GAL4 and UAS::empty (B-I/b-N; UAS::empty/-; FLAG-GAL4/-), or FLAG-GAL4 without a UAS-containing transgene (B-I/b-N; -/-; FLAG-GAL4/-; “no UAS”). In the absence of a UAS-containing transgene, using ChIP to isolate FLAG-GAL4-associated DNA was not expected to capture locus-specific chromatin-interacting proteins, and these plants were used to identify non-specific proteins cross-linked to the FLAG-GAL4 protein or non-b1TR GAL4-associated DNA. After performing ChIP-MS, we found a total of 2432 non-redundant proteins across all samples (min. 1 peptide, 1.2% FDR). Proteins were identified with the antibody isotype control (mouse IgG) for all transgene combinations and these proteins represented less than 13% of all proteins detected (Figure 2A). To take a conservative approach and minimize false positives, we excluded all proteins shared between anti-FLAG and anti-IgG from further analysis, as they could not be confidently distinguished as true UAS::b1TR-interacting proteins. The remaining proteins present in anti-FLAG experiments were separated based on their transgene association, yielding 1461, 1701, and 1553 proteins from UAS::b1TR, UAS::empty, and FLAG-GAL4 (“no UAS”), respectively, with 144 proteins uniquely identified by their association with UAS::b1TR (Figure 2B). To confirm that the protocol captured the FLAG-GAL4 fusion protein, we searched our obtained peptide spectra against the Swissprot reference database and identified the distribution of GAL4-associated peptides. As expected, GAL4 peptides (Uniprot accession P04386) were detected in all biological replicates using antibodies against FLAG while none were detected in any parallel mouse IgG ChIP reactions (Additional Dataset 2). In addition, we performed ChIP-qPCR targeting FLAG-GAL4 with antibodies against FLAG and mouse IgG. Consistent with enrichment by ChIP, we detected higher relative enrichment of the UAS::b1TR transgene sequence when using anti-FLAG compared to mouse IgG (Supplemental Figure S1A).
Although overlap among proteins was observed between transgene combinations, we identified distinct lists of proteins uniquely associated with UAS::b1TR and UAS::empty, providing candidates for further investigation (Figure 2B). We performed Gene Ontology (GO) analysis to identify terms associated with the captured proteins and found significantly enriched Biological Process GO terms (FDR < 0.05) for all anti-FLAG ChIP-MS experiments, with fewer significant terms found for experiments using mouse IgG as expected (Supplemental Figure S1B). Enriched Biological Process GO terms associated with unique UAS::b1TR proteins (FDR ≤ 0.02) include “regulation of gene expression”, “gene silencing”, “regulation of transcription by RNA Polymerase (Pol) II”, “nucleic acid metabolic process”, and “pigment biosynthetic process” (Supplemental Figure S1C). We also found that proteins annotated with the Molecular Function GO term “DNA-binding” were detected only in samples with UAS-containing transgenes, while none were detected in the FLAG-GAL4 alone (no UAS) control (Supplemental Figure S1D). In contrast, RNA-binding proteins showed both shared and distinct distributions across transgenes, with UAS::b1TR-associated proteins uniquely enriched for “double-stranded RNA binding” (Supplemental Figure S1E). These results suggest that our protein lists reflect the expected differences between UAS-containing chromatin and FLAG-GAL4 alone without a cognate UAS-binding site.

3.3. Several RNA-Binding Proteins Are Uniquely Associated with UAS::b1TR Chromatin

While proteins involved in UAS::b1TR regulation may act at multiple locations in the genome and may therefore be associated with the genomic location of any integrated transgene, the proteins unique to the UAS::b1TR are strong candidates for regulatory proteins acting specifically at the transgene locus. Gene ontology analysis was used to identify the predicted function of the 144 putative UAS::b1TR-interacting proteins (Figure 2B). Of the 95 out of 144 UAS::b1TR-associated proteins with annotated GO terms, 9 were classified as RNA-binding (Table 2). Several of these proteins contain features that may indicate a role in RNA processing. One is a predicted RNAse III domain-containing protein (Zm00001d012824) closely related to Arabidopsis thaliana NUCLEAR FUSION DEFECTIVE 2 (Nfd2), which functions in small RNA production through double-stranded RNA (dsRNA) binding. Two DEAD-box RNA helicases, DEAD-BOX RNA HELICASE 53 (ATRH53, Zm00001d041480), and EUKARYOTIC INITIATION FACTOR 4A-2 (EIF4A2, Zm00001d015251), were uniquely detected with UAS::b1TR and contain helicase domains that could facilitate dsRNA processing. Additionally, POLYADENYLATE-BINDING PROTEIN 4 (PAB4, Zm00001d019824) encodes four RNA RECOGNITION MOTIF (RRM) domains, a feature associated with proteins acting in co-transcriptional RNA processing. Other RNA-binding proteins, including the RRM-domain containing HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN A3-LIKE PROTEIN 2 (HNRP3A, Zm00001d044469) and NUCLEAR TRANSPORT FACTOR 2 (NTF2, Zm00001d016528), were shared between the UAS::b1TR and UAS::empty transgenes (Table 2). While the functional relevance of these proteins in the context of UAS::b1TR remains unclear, their predicted activities are consistent with potential roles in co-transcriptional RNA processing.

3.4. Two Sequence-Specific DNA-Binding Proteins Were Uniquely Associated with UAS::b1TR Chromatin and Can Activate GAL Expression in Yeast One-Hybrid

In our analysis, two proteins encoding DNA binding domains were among the 144 found to be unique to UAS::b1TR (Table 3). AGAMOUS-LIKE MADS-BOX PROTEIN AGL5/ZEA AGAMOUS PROTEIN 4 (ZAG4; Zm00001eb120710) is a predicted sequence-specific transcription factor that may be associated with the RNA Pol II transcription of b1TR. ZAG4 is part of a highly-conserved gene family involved in floral homeotic gene expression [42] and is highly expressed in ear and husk leaves. We also identified DDT-TRANSCRIPTION FACTOR 4 (DDT4; Zm00001d022417) which is a 191 kDa protein of unknown function containing two AT-hook DNA binding domains, one DDT domain, and three PHD domains (Supplemental Figure S4A).
We performed cis-regulatory sequence analysis with a single 853 bp unit of the b1TR to determine if motifs suggestive of DDT4 or ZAG4 binding were present. While we did not detect putative ZAG4 binding sites, we detected several potential DDT4 binding sites throughout the b1TR, with most motifs positioned in the first 450 bp (Plant Pan 2.0, similarity score ≥ 0.91; Supplemental Table S4). AT-hook binding motifs were the most prevalent TF binding motif detected throughout the 853 bp unit, covering 18% of the sequence (Supplemental Figure S5). The predicted AT-hook binding motifs overlapped with experimentally-defined functional sequences within the b1TR including the CXC-DOMAIN CONTAINING B1-BINDING PROTEIN (CBBP) binding region [35], predicted yeast autonomous replicating sequences (ARS), and matrix attachment regions [14]. Genome-wide characterization of distal cis-elements in maize has identified several conserved distal element sequence motifs [43], one of which was detected at four sites within the 853 bp unit, with three out of four of these motifs overlapping with AT-hook binding motifs.
To assess whether the two DNA-binding proteins isolated from UAS::b1TR interact with b1TR in a heterologous system, we performed a quantitative yeast one-hybrid (Y1H) assay, which measures β-galactosidase activity as an indicator of transient interactions between proteins and target DNA sequences. In this assay, a plasmid containing the DNA sequence of interest (bait) and a reporter gene is transformed into yeast alongside another plasmid encoding the protein of interest (prey) fused to a GAL4 activation domain (AD). If the prey protein binds to the bait sequence, the GAL4 AD will drive reporter gene expression [37]. In this study, an 853 bp unit of the b1TR was used as bait alongside a LacZ reporter, with CBBP, DDT4, ZAG4, and ZAG4 with its DNA binding domain deleted (ZAG4ΔBD) as the prey (Supplemental Figure S2). As expected, the positive control prey CBBP, a protein shown to strongly activate GAL with b1TR bait [35], activated GAL expression when using an 853 bp unit of b1TR as bait (Figure 2C). Both DDT4 and ZAG4 activated GAL expression with b1TR bait, while no activation was observed in non-b1TR bait controls or when the ZAG4 DNA binding domain was deleted (Figure 2D,E). These results indicate that DDT4 and ZAG4 can activate GAL transcription in a b1TR-specific manner and provide supporting evidence for their association with UAS::b1TR-chromatin detected by ChIP-MS.

3.5. Predicted Interaction Networks of DDT4 and ZAG4 Suggest Chromatin-Associated Functions

We analyzed co-expression networks and protein–protein interaction predictions to explore the potential functions of UAS::b1TR-associated proteins, though additional experimental validation is needed to confirm their roles at this locus. STRING analysis [40] identified ZAG4 as a predicted interactor of AGAMOUS-LIKE MADS-BOX PROTEIN AGL6 (Zm00001d051465; Supplemental Figure S3A), a master regulator of floral development that recruits PRC2 for H3K27me3 deposition in Arabidopsis [44]. Expanding this network revealed additional predicted interactions with maize orthologs of FLOWERING LOCUS D (FLD, Zm00001d002684) and an RNA-RECOGNITION MOTIF (RRM) domain-containing protein (Zm00001d005281), the putative maize ortholog of FLOWERING CONTROL LOCUS A (FCA) (Supplemental Figure S3B). Although FCA was not detected in our ChIP-MS dataset, we detected several RRM-domain-containing proteins (PAB4, NTF2, and HRNPA3) associated with UAS::b1TR (Table 2).
DDT4 contains AT-hook DNA binding domains and PHD domains, which in other systems mediate chromatin interactions. DDT4 displays high amino acid similarity to human BROMODOMAIN PHD TRANSCRIPTION FACTOR (BPTF), which binds H3K4me3 and participates in chromatin remodeling (blastp score = 92.0, e-value = 9 × 10−18; Supplemental Figure S4B,C). Co-expression analysis using a maize gene expression network [39] identified associations with genes involved in chromatin modification, including SET DOMAIN GENE 127 (SDG127; GRMZM2G473138), the maize ortholog of ATSDG25/ATRX7 (Table 4), which regulates H3K4 demethylation in Arabidopsis [45]. DDT4 was also co-expressed with MEDIATOR OF RNA-POLYMERASE II TRANSCRIPTION SUBUNIT 14 (MED14; GRMZM2G446872; Table 4). Our proteomic analysis further detected another mediator complex subunit, HSP6, and the largest Pol II subunit, NRPB1, at UAS::b1TR (Table 3). STRING analysis predicted interactions between DDT4 and several major chromatin regulators, including H3K4-specific methyltransferases SET102 and ASHH1, as well as the ISWI ATPase CHR11, homologous to PICKLE/CHD3 (Table 5). DDT4 was also predicted to interact with CHR101 and CHR106 (Table 5), maize orthologs of DDM1, which maintain DNA methylation and epigenetic silencing [46,47,48,49]. In maize, DDM1 interacts with SHH2 and the RdDM machinery to maintain asymmetric DNA methylation and silence transposable elements [50].
An expanded STRING network incorporating DDT4 and its predicted primary interactors suggest DDT4 interacts broadly with chromatin regulators, chromosome cohesion proteins, and DNA replication factors (Supplemental Table S1). Markov clustering (MCL) (inflation parameter = 7) generated three clusters associated with epigenetic regulation, sister chromatid cohesion, and SMC-related proteins (Figure 2E). Overall, our identification of DDT4 and ZAG4 at UAS::b1TR, and their potential roles in transcriptional regulation, chromatin modification, and DNA replication may provide some insight into their involvement in the epigenetic silencing of b1; however, additional experiments are necessary to confirm their interactions and define their precise role at b1TR.

4. Discussion

Discovery-based proteomics experiments of complex mixtures can identify several thousand proteins, including many non-specific interactions, making interpretation challenging and requiring investigators to manually screen large datasets (reviewed in [51]). To attempt to overcome this, we used genetic controls (UAS::empty and no UAS) alongside UAS::b1TR to identify non-specific proteins that overlap between samples, reducing the effective search space for proteins of interest. The conservative filtering approach resulted in the identification of only two proteins with DNA binding domains, DDT4 and ZAG4, both of which interact with b1TR to drive GAL expression in yeast one-hybrid.
Consistent with our expectations of RNA Pol II transcription of b1TR and the transcriptional silencing associated with UAS::b1TR observed in this study, several of the proteins detected included RNA Pol II subunits, components of Mediator, and were involved in co-transcriptional RNA processing [15,22,29,52]. However, we did not detect CBBP, a CXC-domain containing zinc finger transcription factor that interacts with b1TR [35]. CBBP strongly activates GAL expression with b1TR bait in the yeast one-hybrid assay and is sufficient to induce silencing and paramutation when overexpressed [35]. The absence of CBBP in our ChIP-MS data could reflect inherent limitations in the technique or native tissue-specific expression of CBBP. CBBP (also known as CPP2; GRMZM2G015097) is expressed in seed and is low or not expressed in sheath tissue and may therefore not be interacting at b1TR in our samples (Supplemental Figure S6). This may also represent differences in the proteins involved in the establishment and maintenance stages of paramutation which occur during reproduction and vegetative growth, respectively [53]. Despite CBBP not being detected in our dataset, this does not compromise the overall conclusions of the study, which are based on factors active during the maintenance phase in vegetative tissue.
One of the few differences between b1TR in B′ versus B-I is the enrichment of H3K9me2 and H3K27me2 at silenced b1TR and H3K9ac at active b1TR [16,54]. Sequence-specific DNA binding proteins were expected to play an important role in b1TR-mediated silencing, enhancement, and paramutation of b1 because of the local enrichment of transcription factor binding motifs present in the hepta-repeat relative to accessions with only one 853 bp copy, for example B73, which does not participate in paramutation or display enhancement of b1 [13,14,29]. The requirement for repetitive motifs may be involved in higher-order oligomerization of transcription factors in three-dimensional chromatin organization [55], and may explain the lower GAL activation by DDT4 and ZAG4 relative to CBBP in our yeast one-hybrid assay, which did not include endogenous chromatin context, accessory factors, or seven tandem copies of b1TR.
The first PHD domain of DDT4 displayed high amino acid sequence conservation to the first PHD domain of human BPTF which binds H3K4me3 at actively transcribed loci ([56], reviewed in [57]). ZAG4 was predicted to interact with AGL6 and the maize ortholog of FLOWERING LOCUS D which, in Arabidopsis, recruits H3K4-specific demethylases and histone deacetylases HDAC5/6 to Flowering locus C (FLC) [58,59]. Despite the absence of the canonical MADS-box/SRF/MCM “CArG” (CC(A/T)6GG) recognition motif in b1TR, slight modifications to the consensus were tolerated with reduced affinity for AGAMOUS (AG) binding and this affinity varied depending on whether AG formed a homo or heterodimer with related AGs [60,61]. ZAG4 and DDT4 were predicted to interact with several maize H3K4 methyltransferases which may work to regulate Pol II transcription of b1TR and affect siRNA levels [52], leading to an increase in 24-nt siRNA biogenesis from UAS::b1TR, triggering cis and trans silencing of b1TR. This is exemplified by the copy number-dependent transition of 21-nt to 24-nt siRNA in de novo transposon silencing of the active EVADE retrotransposon by RdDM, which requires a threshold of ~40 copies before fully penetrant silencing is triggered [62,63].
Proteins related to co-transcriptional RNA processing and small RNA biogenesis were identified uniquely with UAS::b1TR, for example, the RNAse III domain containing protein NFD2 which interacted directly with 21-nt siRNA-producing DICER-LIKE 4 in Arabidopsis thaliana [64]. PAB4, HNRPA3, and NTF2 are RNA RECOGNITION MOTIF (RRM)-domain-containing proteins associated with UAS::b1TR. RRM domains are ubiquitous protein domains across eukaryotes that bind single stranded RNA and are associated with post-transcriptional gene regulation [65]. Although RRM-domain proteins were also detected in UAS::empty samples, we focused on the functional relevance of specific RNA-binding proteins uniquely associated with UAS::b1TR rather than on domain frequency alone. In Arabidopsis, the RRM-domain containing protein FLOWERING TIME CONTROL PROTEIN A (FCA) is required for the deposition of asymmetric DNA methylation at single/low copy repeat loci [66], and the maize FCA ortholog was predicted to interact with FLD, ZAG4 and AGL6. We also found HLN and PAI1 uniquely associated with UAS::b1TR. In humans, these proteins interact directly with CHD3 [67] and are involved in mRNA stability [68,69]. The Drosophila melanogaster orthologs of HLN and PAI1 and VIG and VIG2 are components of the RNA-induced silencing complex [70] that are involved in the inheritance of heterochromatin [71] and they have been shown to interact directly with the actively transcribed chromatin of the Histone C gene cluster [12]. These examples suggest that these RNA binding proteins may be important in co-transcriptional RNA processing and the recruitment of H3K4 demethylases and HDACs to establish transcriptional gene silencing of b1TR.
In this study, and to our knowledge for the first time in plants, we identified in vivo the chromatin-interacting proteins of a stably integrated transgene locus. The proteins that interact with UAS::b1TR chromatin may be responsible for the dramatic phenotypic differences between transcriptional enhancement and the silencing of b1 between epi-alleles. Our approach has identified subunits of RNA Pol II, RNA-binding proteins, and chromatin-interacting proteins associated with UAS::b1TR, which may be involved in transgene-mediated trans-silencing of endogenous b1TR. In addition, we detected and confirmed sequence specificity through GAL activation of two unique UAS::b1TR-associated proteins, ZAG4 and DDT4. Subsequent genetic studies of these proteins will be critical to determine the mechanism for conferring the active versus silenced state of the b1TR in B-I and B′, respectively. In addition, the use of CRISPR-based ChIP-MS and proximity labeling approaches can improve our ability to identify and characterize the consortia of trans factors interacting with the b1TR and other cis regulatory loci, an important step towards efforts deciphering their effects on gene expression and inheritance.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14121863/s1, Figure S1: (A) Enrichment of UAS::b1TR chromatin fragments using anti-FLAG versus anti-mouse IgG detected by ChIP-qPCR. (B) Comparison of the number of enriched (FDR < 0.05) GO terms between transgenes and control ChIP-MS experiments using mouse IgG. (C) Shared and overlapping significantly enriched (FDR < 0.05) Biological Process GO terms between UAS::b1TR and UAS::empty. (D) Percentage of GO terms within the DNA Binding Proteins category unique to each transgene. Samples from plants that were not transgenic for a UAS-containing transgene (no UAS) did not capture DNA binding proteins. (E) Percentage of GO terms within the RNA Binding Proteins category unique to each transgene; Figure S2: (A) Preparation of transgenic yeast strains. (B) Quantitative yeast one-hybrid assay; Figure S3: (A) Predicted protein-protein interactions of ZAG4 showing primary interaction between ZAG4 and AGL6. (B) Expanded protein-protein interaction network using ZAG4 and AGL6 as anchor interactors. (C) Gene co-expression network analysis focused on DDT4. (D) STRING protein-protein interaction (PPI) network focused on DDT4; Figure S4: Visualization of domains and local homology of maize DDT4 and human BPTF. (A) SMART domain annotation of the maize DDT4 protein. (B) SMART domain annotation of the human BPTF protein. (C) Pair-wise amino acid sequence alignment between DDT4 and BPTF showing conservation between the DDT domains and first PHD domains made by Jalview; Figure S5: Annotation of b1TR sequence features; Figure S6: Gene expression atlas showing tissue specific expression of CBBP/CPP2 (GRMZM2G015097) mainly in seed and low/no expression in vegetative tissues; Table S1: Expanded protein-protein interactions involving DDT4 primary Protein-protein interactions; Table S2: 144 Proteins uniquely associated with UAS::b1TR; Table S3: Plasmid Constructs and Strains Used in the Yeast Transformation Experiment; Table S4: Transcription factor binding motif analysis of the b1TR.

Author Contributions

Conceived and designed the experiments: K.M.M. and J.S.L. Performed the experiments: J.S.L., K.M.K., Y.B.G. and J.E. Analyzed the data: J.S.L., K.M.K. and Y.B.G. Interpreted results: K.M.M. and J.S.L. Wrote the manuscript: K.M.M. and J.S.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by NSF grant number 22-510-2243562.

Data Availability Statement

Seed for all transgenic and control lines are available upon request. Proteomic data from all experiments used for this analysis was exported from Scaffold and is available in Additional Dataset 1 and Additional Dataset 2 and data from yeast one-hybrid experiments is available in Additional Dataset 3.

Acknowledgments

We thank Vicki Chandler, Jan Brzeski, and Kasia Brzeska for generating and providing the UAS::b1TR, UAS::empty, and FLAG-GAL4 plasmids and seed stocks. We thank Phebe Lemert, Curt Martinez, Amlex Bencosme, and Julia Won for their assistance in the field pigmentation scoring experiments and cloning. We thank Rakesh Singh and Cindy Vied (Florida State University College of Medicine) for technical advice and mass-spectrometry services. We thank Hong-Guo Yu for supplying equipment used in the Y1H and for his expertise in yeast systems, as well as Lauren Carson and Anuvind Kalpetta Gramathil. We also thank Cheryl Pye, Brian Washburn, and Diego Zorio from the FSU Biology Core Facilities for their expertise and help with cloning. We thank Jonathan Dennis for critical advice and fruitful discussion. ChatGPT 4o was used for assistance with writing at the final stage of manuscript preparation.

Conflicts of Interest

The authors declare no conflict of interest.

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Plants 14 01863 g001
Figure 1. Pigmentation phenotypes of plants transgenic for UAS::b1TR and UAS::empty transgenes. (A) Schematic of the endogenous b1TR enhancer (red dashed line designates long-range chromatin loops that form with b1), and UAS::b1TR, UAS::empty, and FLAG-GAL4 transgenes. The UAS::b1TR transgene encodes identical hepta repeats (grey boxes) cloned from b1TR fused to an adjacent 10× GAL4-binding upstream activation sequence array (UAS; blue boxes) providing unique binding sites for FLAG-GAL4. (B) Representative phenotypes of relevant b1 genotypes (B-I silenced (B’) or B-I active) of non-transgenic plants and plants transgenic for either UAS::b1TR or UAS::empty. (C) Crossing scheme and expected sheath pigmentation with or without silencing for offspring segregating for a transgene (UAS::b1TR or UAS::empty) and b1 alleles. (D) Percentage of light plants across all segregating families of transgenic and non-transgenic siblings and controls. White dashed line indicates expected proportion of light plants without B-I silencing.
Figure 1. Pigmentation phenotypes of plants transgenic for UAS::b1TR and UAS::empty transgenes. (A) Schematic of the endogenous b1TR enhancer (red dashed line designates long-range chromatin loops that form with b1), and UAS::b1TR, UAS::empty, and FLAG-GAL4 transgenes. The UAS::b1TR transgene encodes identical hepta repeats (grey boxes) cloned from b1TR fused to an adjacent 10× GAL4-binding upstream activation sequence array (UAS; blue boxes) providing unique binding sites for FLAG-GAL4. (B) Representative phenotypes of relevant b1 genotypes (B-I silenced (B’) or B-I active) of non-transgenic plants and plants transgenic for either UAS::b1TR or UAS::empty. (C) Crossing scheme and expected sheath pigmentation with or without silencing for offspring segregating for a transgene (UAS::b1TR or UAS::empty) and b1 alleles. (D) Percentage of light plants across all segregating families of transgenic and non-transgenic siblings and controls. White dashed line indicates expected proportion of light plants without B-I silencing.
Plants 14 01863 g001
Plants 14 01863 g002
Figure 2. Results from UAS:transgene-targeted ChIP-MS, quantitative yeast one-hybrid assay results, and DDT4 interaction analysis (A) Average number of proteins identified by ChIP-MS for plants transgenic for the FLAG-GAL4 transgene without a UAS transgene (no UAS), or with a bait transgene (UAS::b1TR or UAS::empty). Error bars show the standard deviation for number of proteins between two biological replicates. (B) Distribution of identified proteins between bait transgenes from FLAG-GAL4 ChIP-MS experiments. (C) Beta-galactosidase expression was calculated based on absorbance values for b1TR or non-b1TR negative control bait and either CBBP b1TR binding protein prey (positive control), (D) DDT4 prey, or (E) ZAG4 full length or ZAG4ΔBD (without DNA binding domain) prey. (F) Expanded STRING protein–protein interaction network and MCL clustering with DDT4 primary interactors. The primary interactors of the DDT4-interacting protein set are shown as colored nodes. Secondary and tertiary interactions are shown as grey nodes. MCL clusters were manually annotated based on constituent proteins of each cluster. Nodes are connected by edges (lines) which are colored to indicate the interaction. The black arrow points to the location of DDT4 in the network.
Figure 2. Results from UAS:transgene-targeted ChIP-MS, quantitative yeast one-hybrid assay results, and DDT4 interaction analysis (A) Average number of proteins identified by ChIP-MS for plants transgenic for the FLAG-GAL4 transgene without a UAS transgene (no UAS), or with a bait transgene (UAS::b1TR or UAS::empty). Error bars show the standard deviation for number of proteins between two biological replicates. (B) Distribution of identified proteins between bait transgenes from FLAG-GAL4 ChIP-MS experiments. (C) Beta-galactosidase expression was calculated based on absorbance values for b1TR or non-b1TR negative control bait and either CBBP b1TR binding protein prey (positive control), (D) DDT4 prey, or (E) ZAG4 full length or ZAG4ΔBD (without DNA binding domain) prey. (F) Expanded STRING protein–protein interaction network and MCL clustering with DDT4 primary interactors. The primary interactors of the DDT4-interacting protein set are shown as colored nodes. Secondary and tertiary interactions are shown as grey nodes. MCL clusters were manually annotated based on constituent proteins of each cluster. Nodes are connected by edges (lines) which are colored to indicate the interaction. The black arrow points to the location of DDT4 in the network.
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Table 1. Results of sheath pigmentation scoring for transgenic lines.
Table 1. Results of sheath pigmentation scoring for transgenic lines.
GenotypeNo. PlantExp. LightObs. LightExp. DarkObs. Darkχ2p Value
UAS::b1TR/-1791791610181.810.1785
UAS::empty/-16716710805920.844.98 × 10−6
Chi square test (χ2) was performed with phenotypic data for plants transgenic for UAS::b1TR or UAS::empty to estimate the degree of confidence for the null hypothesis of 1:0 light–dark plants, which would be expected if the transgene silenced B-I.
Table 2. Selected RNA-interacting proteins associated with the UAS::b1TR.
Table 2. Selected RNA-interacting proteins associated with the UAS::b1TR.
Protein NameGene IDAccessionPredicted Function (GO)
Hyaluronan/mRNA binding family *HlnB4FR90RNA-binding
Plasminogen activator inhibitor 1 RNA-binding protein *Pai1A0A1D6FLF3RNA-binding
Polyadenylate-binding protein *Pab4A0A1D6I0G0RNA-binding
Nuclear fusion defective 2 *Nfd2B6SGY5production of siRNA in RNA-interference
DEAD-box ATP-dependent RNA helicase 53 *Atrh53A0A1D6MWD6helicase activity
Eukaryotic initiation factor 4A-2 *Eif4a-2B4FBK3helicase activity
Heterogeneous nuclear ribonucleoprotein A3-like protein 2 +Hnrpa3A0A096QLU9mRNA processing
Nuclear transport factor 2 +Ntf2A0A1D6H8Q2RNA binding
THO complex subunit 4 ++Tho4B6T346RNA-binding
Ribonuclease (Tudor-SN1) ++Tsn1K7UVD7gene silencing by RNA
* Proteins uniquely identified with UAS::b1TR. + Proteins shared between UAS::b1TR and UAS::empty. ++ Proteins shared between UAS::b1TR, UAS::empty, and no-UAS.
Table 3. Selected DNA-interacting and chromatin-interacting proteins associated with UAS::b1TR.
Table 3. Selected DNA-interacting and chromatin-interacting proteins associated with UAS::b1TR.
Protein NameGene IDAccessionPredicted Function (GO)
DDT-transcription factor 4 *Ddt4A0A096S3Y8metal ion binding
Agamous-like MADS-box protein AGL5 *Agl5/Zag4A0A1D6MHD4DNA-binding transcription factor activity
DNA repair protein RAD23 +Rad23B6TB61nucleotide-excision repair
Putative AP2/EREBP transcription factor superfamily protein ++Ereb186A0A1D6FGH1DNA-binding transcription factor activity
RNA_pol_Rpb1_2 domain-containing protein +Nrpb1A0A1x7YI76DNA-directed 5′-3′ RNA polymerase activity
Putative mediator of RNA polymerase II transcription subunit 37c ++Hsp6A0A1D6N7I4ATPase activity
Actin-related protein 4 ++Arp4B4FNB4regulation of transcription by RNA polymerase II
Regulator of chromosome condensation 2 ++Rcc2B4FTT2chromatin binding
Nucleosome assembly protein 1 ++Nfa102A0A1D6FWR5nucleosome assembly
Histone deacetylase complex subunit SAP18 ++Sap18B4FIB4
* Proteins uniquely identified with UAS::b1TR. + Proteins shared between UAS::b1TR and UAS::empty. ++ Proteins shared between UAS::b1TR, UAS::empty, and no-UAS.
Table 4. Ddt4 gene co-expression network analysis showing primary nodes.
Table 4. Ddt4 gene co-expression network analysis showing primary nodes.
Gene NameGene IDPredicted FunctionE-ValueScore
Chr5GRMZM2G010342chromatin assembly or disassembly060.52
Mybr85GRMZM2G089406RNA splicing3.00 × 10−9931.28
Fab1bGRMZM2G111208 055.19
Btaf1GRMZM2G168096nucleic acid binding060.88
Sin3-like 4GRMZM2G334457regulation of transcription051.44
Ptm3GRMZM2G403562regulation of transcription3.00 × 10−13833.65
Med14GRMZM2G446872positive regulation of transcription053.2
Sdg127GRMZM2G473138histone lysine methylation2.00 × 10−9869.78
Table 5. STRING primary protein–protein interactions of maize DDT4.
Table 5. STRING primary protein–protein interactions of maize DDT4.
Protein NameGene IDCo-ExpressionExp. Defined InteractionText MiningCombined Score
Histone methyltransferase (H3-K4 specific)GRMZM2G35243100.7190.7180.917
Uncharacterized proteinZm00001d0366010.83200.2090.861
Histone-lysine N-methyltransferase ASHH1GRMZM2G1476190.83200.1850.857
Sister chromatid cohesion proteinZm00001d0186570.83200.1850.857
Sister chromatid cohesion proteinZm00001d0079430.83200.1850.857
Chromatin complex subunit A101GRMZM2G1771650.0590.6570.4580.809
Chromatin complex subunit A106GRMZM2G0710250.1290.1610.360.491
Putative helicase CHR10GRMZM2G0491680.1290.1610.360.491
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Lynn, J.S.; Koirtyohann, K.M.; Gebreab, Y.B.; Edwards, J.; McGinnis, K.M. Identification of Proteins Associated with Stably Integrated Maize b1 Tandem Repeat Transgene Chromatin. Plants 2025, 14, 1863. https://doi.org/10.3390/plants14121863

AMA Style

Lynn JS, Koirtyohann KM, Gebreab YB, Edwards J, McGinnis KM. Identification of Proteins Associated with Stably Integrated Maize b1 Tandem Repeat Transgene Chromatin. Plants. 2025; 14(12):1863. https://doi.org/10.3390/plants14121863

Chicago/Turabian Style

Lynn, Jason S., Kathryn M. Koirtyohann, Yacob B. Gebreab, Jaliyah Edwards, and Karen M. McGinnis. 2025. "Identification of Proteins Associated with Stably Integrated Maize b1 Tandem Repeat Transgene Chromatin" Plants 14, no. 12: 1863. https://doi.org/10.3390/plants14121863

APA Style

Lynn, J. S., Koirtyohann, K. M., Gebreab, Y. B., Edwards, J., & McGinnis, K. M. (2025). Identification of Proteins Associated with Stably Integrated Maize b1 Tandem Repeat Transgene Chromatin. Plants, 14(12), 1863. https://doi.org/10.3390/plants14121863

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