Genomic Binding Sites Research Articles

Identifying transcription factors with cell-type specific DNA binding signatures

BackgroundTranscription factors (TFs) bind to different parts of the genome in different types of cells, but it is usually assumed that the inherent DNA-binding preferences of a TF are invariant to cell type. Yet, there are several known examples of TFs that switch their DNA-binding preferences in different cell types, and yet more examples of other mechanisms, such as steric hindrance or cooperative binding, that may result in a “DNA signature” of differential binding.ResultsTo survey this phenomenon systematically, we developed a deep learning method we call SigTFB (Signatures of TF Binding) to detect and quantify cell-type specificity in a TF’s known genomic binding sites. We used ENCODE ChIP-seq data to conduct a wide scale investigation of 169 distinct TFs in up to 14 distinct cell types. SigTFB detected statistically significant DNA binding signatures in approximately two-thirds of TFs, far more than might have been expected from the relatively sparse evidence in prior literature. We found that the presence or absence of a cell-type specific DNA binding signature is distinct from, and indeed largely uncorrelated to, the degree of overlap between ChIP-seq peaks in different cell types, and tended to arise by two mechanisms: using established motifs in different frequencies, and by selective inclusion of motifs for distint TFs.ConclusionsWhile recent results have highlighted cell state features such as chromatin accessibility and gene expression in predicting TF binding, our results emphasize that, for some TFs, the DNA sequences of the binding sites contain substantial cell-type specific motifs.

CRISPR/Cas13 sgRNA-Mediated RNA-RNA Interaction Mapping in Live Cells with APOBEC RNA Editing.

Current research on long non-coding RNA (lncRNA) has predominantly focused on identifying their protein partners and genomic binding sites, leaving their RNA partners largely unknown. To address this gap, the study has developed a method called sarID (sgRNA scaffold assisted RNA-RNA interaction detection), which integrates Cas13-based RNA targeting, sgRNA engineering, and proximity RNA editing to investigate lncRNA-RNA interactomes. By applying sarID to the lncRNA NEAT1, over one thousand previously unidentified binding transcripts are discovered. sarID is further expanded to investigate binders of XIST, MALAT1, NBR2, and DANCR, demonstrating its broad applicability in identifying lncRNA-RNA interactions. The findings suggest that lncRNAs may regulate gene expression by interacting with mRNAs, expanding their roles beyond known functions as protein scaffolds, miRNA sponges, or guides for epigenetic modulators. sarID has the potential to be adapted for studying other specific RNAs, providing a novel immunoprecipitation-free method for uncovering RNA partners and facilitating the exploration of the RNA-RNA interactome.

P53motifDB: integration of genomic information and tumor suppressor p53 binding motifs.

The tumor suppressor gene TP53 encodes the DNA binding transcription factor p53 and is one of the most commonly mutated genes in human cancer. Tumor suppressor activity requires binding of p53 to its DNA response elements and subsequent transcriptional activation of a diverse set of target genes. Despite decades of close study, the logic underlying p53 interactions with its numerous potential genomic binding sites and target genes is not yet fully understood. Here, we present a database of DNA and chromatin-based information focused on putative p53 binding sites in the human genome to allow users to generate and test new hypotheses related to p53 activity in the genome. Users can query genomic locations based on experimentally observed p53 binding, regulatory element activity, genetic variation, evolutionary conservation, chromatin modification state, and chromatin structure. We present multiple use cases demonstrating the utility of this database for generating novel biological hypotheses, such as chromatin-based determinants of p53 binding and potential cell type-specific p53 activity. All database information is also available as a precompiled sqlite database for use in local analysis or as a Shiny web application.

Pharmacological restriction of genomic binding sites redirects PU.1 pioneer transcription factor activity

Transcription factor (TF) DNA-binding dynamics govern cell fate and identity. However, our ability to pharmacologically control TF localization is limited. Here we leverage chemically driven binding site restriction leading to robust and DNA-sequence-specific redistribution of PU.1, a pioneer TF pertinent to many hematopoietic malignancies. Through an innovative technique, ‘CLICK-on-CUT&Tag’, we characterize the hierarchy of de novo PU.1 motifs, predicting occupancy in the PU.1 cistrome under binding site restriction. Temporal and single-molecule studies of binding site restriction uncover the pioneering dynamics of native PU.1 and identify the paradoxical activation of an alternate target gene set driven by PU.1 localization to second-tier binding sites. These transcriptional changes were corroborated by genetic blockade and site-specific reporter assays. Binding site restriction and subsequent PU.1 network rewiring causes primary human leukemia cells to differentiate. In summary, pharmacologically induced TF redistribution can be harnessed to govern TF localization, actuate alternate gene networks and direct cell fate.

Chemical restriction of PU.1 genomic binding sites activates alternate gene networks.

Chemical restriction of PU.1 genomic binding sites activates alternate gene networks.

Injury-induced Foxm1 expression in the mouse kidney drives epithelial proliferation by a cyclin F-dependent mechanism.

Acute kidney injury (AKI) strongly upregulates the transcription factor Foxm1 in the proximal tubule in vivo, and Foxm1 drives epithelial proliferation in vitro. Here, we report that deletion of Foxm1 either with a nephron-specific Cre driver or by inducible global deletion reduced proximal tubule proliferation after ischemic injury in vivo. Foxm1 deletion led to increased AKI to chronic kidney disease transition, with enhanced fibrosis and ongoing tubule injury 6 weeks after injury. We report ERK mediated FOXM1 induction downstream of the EGFR in primary proximal tubule cells. We defined FOXM1 genomic binding sites by cleavage under targets and release using nuclease (CUT&RUN) and compared the genes located near FOXM1 binding sites with genes downregulated in primary proximal tubule cells after FOXM1 knockdown. The aligned data sets revealed the cell cycle regulator cyclin F (CCNF) as a putative FOXM1 target. We identified 2 cis regulatory elements that bound FOXM1 and regulated CCNF expression, demonstrating that Ccnf is strongly induced after kidney injury and that Foxm1 deletion abrogates Ccnf expression in vivo and in vitro. Knockdown of CCNF also reduced proximal tubule proliferation in vitro. These studies identify an ERK/FOXM1/CCNF signaling pathway that regulates injury-induced proximal tubule cell proliferation.

Disordered sequences of transcription factors regulate genomic binding by integrating diverse sequence grammars and interaction types.

Intrinsically disordered regions (IDRs) guide transcription factors (TFs) to their genomic binding sites, raising the question of how structure-lacking regions encode for complex binding patterns. We investigated this using the TF Gln3, revealing sets of IDR-embedded determinants that direct Gln3 binding to respective groups of functionally related promoters, and enable tuning binding preferences between environmental conditions, phospho-mimicking mutations, and orthologs. Through targeted mutations, we defined the role of short linear motifs (SLiMs) and co-binding TFs (Hap2) in stabilizing Gln3 at respiration-chain promoters, while providing evidence that Gln3 binding at nitrogen-associated promoters is encoded by the IDR amino-acid composition, independent of SLiMs or co-binding TFs. Therefore, despite their apparent simplicity, TF IDRs can direct and regulate complex genomic binding patterns through a combination of SLiM-mediated and composition-encoded interactions.

Regulation and activity of Runx2 in CD8+ T cells responding to infection promotes long-term survival

Abstract Constitutive expression of Runx family proteins in CD8 T cells and maintenance of accessibility surrounding Runx motifs in regulatory regions of lineage-specific loci suggest that they may play a role in establishing and maintaining the epigenetic state of CD8 T cells. Additionally, Runx proteins possess a highly homologous DNA binding domain, leading not only to redundancies, but also potential competition between Runx proteins for the same genomic binding sites. While Runx1 and Runx3 have well known roles in the development of CD8 T cells, the function of Runx2 is not well established. To further explore the function of Runx2, our lab utilizes a model of retroviral overexpression and CRISPR knockout of Runx proteins in OT-1 CD8 T cells. Adoptive transfer of Runx2 overexpressing or knockout cells followed by ovalbumin-expressing Listeria monocytogenes (LmOva) infection suggests that Runx2 activity is important for the expression and maintenance of genes needed for long-term survival and function, such as Tcf7 and Il7ra. Data analyzed from the literature and chromatin analyses from our lab suggest that Runx proteins may compete for binding sites and drive opposing transcriptional and epigenetic profiles in CD8 T cells after infection. Preliminary data also give insight into the mechanisms controlling Runx2 expression, suggesting that the signals received during priming may affect the balance of Runx protein activity.

PML::RARA and GATA2 proteins interact via DNA templates to induce aberrant self-renewal in mouse and human hematopoietic cells

The underlying mechanism(s) by which the PML::RARA fusion protein initiates acute promyelocytic leukemia is not yet clear. We defined the genomic binding sites of PML::RARA in primary mouse and human hematopoietic progenitor cells with V5-tagged PML::RARA, using anti-V5-PML::RARA chromatin immunoprecipitation sequencing and CUT&RUN approaches. Most genomic PML::RARA binding sites were found in regions that were already chromatin-accessible (defined by ATAC-seq) in unmanipulated, wild-type promyelocytes, suggesting that these regions are "open" prior to PML::RARA expression. We found that GATA binding motifs, and the direct binding of the chromatin "pioneering factor" GATA2, were significantly enriched near PML::RARA binding sites. Proximity labeling studies revealed that PML::RARA interacts with ~250 proteins in primary mouse hematopoietic cells; GATA2 and 33 others require PML::RARA binding to DNA for the interaction to occur, suggesting that binding to their cognate DNA target motifs may stabilize their interactions. In the absence of PML::RARA, Gata2 overexpression induces many of the same epigenetic and transcriptional changes as PML::RARA. These findings suggested that PML::RARA may indirectly initiate its transcriptional program by activating Gata2 expression: Indeed, we demonstrated that inactivation of Gata2 prior to PML::RARA expression prevented its ability to induce self-renewal. These data suggested that GATA2 binding creates accessible chromatin regions enriched for both GATA and Retinoic Acid Receptor Element motifs, where GATA2 and PML::RARA can potentially bind and interact with each other. In turn, PML::RARA binding to DNA promotes a feed-forward transcriptional program by positively regulating Gata2 expression. Gata2 may therefore be required for PML::RARA to establish its transcriptional program.

Abstract 1704: Interplay of MITF and NCOA3 in orchestrating oncogenic transcriptional network in uveal melanoma

Abstract Uveal melanoma (UM) originates from melanocytes residing in the uveal tract of an eye. Despite effective local control, 50% of patients develop metastatic disease responding poorly to chemotherapy, targeted therapy, and immunotherapy. The molecular basis of events leading to initiation of UM is related to mutations in G alpha proteins GNAQ or GNA11, rendering them constitutively active and activating downstream PKC, ERK and AKT pathways sustaining cell proliferation. In our previous work, we showed that NCOA3 regulates proliferation of UM cells in vitro and the growth of UM xenografts in vivo downstream of GNAQ or GNA11. The critical role of NCOA3 in UM is manifested by transcriptional regulation of proliferation-related genes that are massively suppressed by NCOA3 knockout or knockdown. Here, we interrogated transcription factors that NCOA3 interacts with in UM to execute oncogenic programs and identified Melanocyte inducing transcription factor (MITF) as its critical partner. Chromatin immunoprecipitation (ChIP) using an NCOA3 recognizing antibody followed by sequencing was performed to determine NCOA3 binding sites in the human genome. The known transcription binding motifs within NCOA3 peaks were discovered using HOMER algorithm. Co-immunoprecipitation was performed to examine direct NCOA3 and MITF interaction. ChIP sequencing for MITF was performed to determine co-localization of NCOA3 and MITF at chromatin. Proliferation of UM cells after inhibition of MITF by either siRNA mediated knockdown or MITF inhibitor ML329 was evaluated using MTT assay. Functional enrichment analysis of transcriptome was used to identify classes of genes regulated by MITF, with specific attention to the subset regulated by MITF partnered with NCOA3. NCOA3 chromatin peaks are highly enriched in MITF binding motifs, and 77% of the NCOA3 peaks co-localizes with the MITF peaks. NCOA3 co-immunoprecipitates with MITF. Among transcriptional programs regulated by MITF in UM are those related to melanocyte biology, differentiation, and melanin production, but also those related to melanoma growth, especially cell division, DNA replication, myc response, purine and pyrimidine metabolism, and mTOR signaling. Strong suppression of these transcriptional signatures by inhibition of MITF using siRNA-mediated knockdown or pharmacological inhibition correlates with dramatic loss of cell viability. Clinical data indicate that higher expression of MITF-positively regulated genes correlates with lower survival rates of UM patients. Importantly, the transcriptional programs regulated by MITF cooperating with NCOA3 were related to TGF beta signaling, cell adhesion, ECM interaction and actin cytoskeleton. Importance of MITF as an oncogene is manifested in uveal melanoma cell lines and clinical data. Oncogenic function of MITF is partially dependent on its interaction with a transcription co-activator NCOA3. Citation Format: Aleksandra Rusin, Maria E. Echartea, Sai P. Manikonda, Darlene G. Skapura, Christel M. Davis, Erik A. Ehli, Sandra L. Grimm, Cristian Coarfa, Salma Kaochar. Interplay of MITF and NCOA3 in orchestrating oncogenic transcriptional network in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1704.

Abstract 3584: Context matters: Genomic profiling of PR isoform-specific actions in breast cancer stem cells reveals novel therapeutic insights for ER+ breast cancer

Abstract Exposure to progesterone is a recognized risk factor for breast cancer, and PGR polymorphisms are associated with various malignancies. The two progesterone receptor (PR) isoforms, full length PR-B and truncated PR-A, are expressed from the PGR gene in breast tissue and play crucial roles in normal physiology. An imbalance in the expression ratio of these isoforms, favoring increased levels of PR-A, is common in breast cancer and is associated with resistance to tamoxifen in Luminal A-type tumors. Notably, PRs have recently been implicated in promoting endocrine resistance and driving the expansion of cancer stem-like cell (CSC) populations implicated in early dissemination and maintenance of metastatic populations. Because prior gene expression studies were primarily conducted in 2D culture conditions, the isoform-specific molecular and epigenetic mechanisms underlying PR actions in advanced ER+ breast cancer remain elusive. Our goal was to define progesterone-driven gene expression in CSC cells by employing transcriptomic profiling of T47D cells cultivated in spheroid-culture conditions (3D) expressing exclusively PR-A or PR-B. Utilizing CUT&RUN, we have mapped the genomic binding sites unique to each PR isoform and identified shared sites. This genomic data has been integrated with phenotypic studies in cells expressing a single PR isoform. Our findings indicate that PR-A acts as a regulator of the cell cycle and senescence, while PR-B plays a pivotal role in cell metabolism and intracellular signaling. We find little overlap between PR-regulated gene sets derived from the same hormone-treated cells harvested from 2D relative to 3D conditions. Our genomic profiling of PRs in this 3D model system has unveiled novel strategies to combat CSC outgrowth and late recurrence in luminal breast cancer patients. This work has shifted our prior understanding of the role of PRs in gene regulation within the CSC population, and offers potential insights for therapeutic interventions in ER+ breast cancer. Citation Format: Noelle E. Gillis, Thu H. Truong, Jessica Finlay-Schultz, Carol A. Sartorius, Carol A. Lange. Context matters: Genomic profiling of PR isoform-specific actions in breast cancer stem cells reveals novel therapeutic insights for ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3584.

A progesterone derivative linked to a stable phospholipid activates breast cancer cell response without leaving the cell membrane

In hormone-responsive breast cancer cells, progesterone (P4) has been shown to act via its nuclear receptor (nPR), a ligand-activated transcription factor. A small fraction of progesterone receptor is palmitoylated and anchored to the cell membrane (mbPR) forming a complex with estrogen receptor alpha (ERα). Upon hormone exposure, either directly or via interaction with ERα, mbPR activates the SRC/RAS/ERK kinase pathway leading to phosphorylation of nPR by ERK. Kinase activation is essential for P4 gene regulation, as the ERK and MSK1 kinases are recruited by the nPR to its genomic binding sites and trigger chromatin remodeling. An interesting open question is whether activation of mbPR can result in gene regulation in the absence of ligand binding to intracellular progesterone receptor (iPR). This matter has been investigated in the past using P4 attached to serum albumin, but the attachment is leaky and albumin can be endocytosed and degraded, liberating P4. Here, we propose a more stringent approach to address this issue by ensuring attachment of P4 to the cell membrane via covalent binding to a stable phospholipid. This strategy identifies the actions of P4 independent from hormone binding to iPR. We found that a membrane-attached progestin can activate mbPR, the ERK signaling pathway leading to iPR phosphorylation, initial gene regulation and entry into the cell cycle, in the absence of detectable intracellular progestin.

Genomic transcription factor binding site selection is edited by the chromatin remodeling factor CHD4.

Biologically precise enhancer licensing by lineage-determining transcription factors enables activation of transcripts appropriate to biological demand and prevents deleterious gene activation. This essential process is challenged by the millions of matches to most transcription factor binding motifs present in many eukaryotic genomes, leading to questions about how transcription factors achieve the exquisite specificity required. The importance of chromatin remodeling factors to enhancer activation is highlighted by their frequent mutation in developmental disorders and in cancer. Here, we determine the roles of CHD4 inenhancer licensing and maintenance in breast cancer cells and during cellular reprogramming. In unchallenged basal breast cancer cells, CHD4 modulates chromatin accessibility. Its depletion leads to redistribution of transcription factors to previously unoccupied sites. During cellular reprogramming induced by the pioneer factor GATA3, CHD4 activity is necessary to prevent inappropriate chromatin opening. Mechanistically, CHD4 promotes nucleosome positioning over GATA3 binding motifs to compete with transcription factor-DNA interaction. We propose that CHD4 acts as a chromatin proof-reading enzyme that prevents unnecessary gene expression by editing chromatin binding activities of transcription factors.

Molecular Basis of Cell Reprogramming into iPSCs with Exogenous Transcription Factors.

A striking discovery in recent decades concerning the transcription factor (TF)-dependent process was the production of induced pluripotent stem cell (iPSCs) from fibroblasts by the exogenous expression of the TF cocktail containing Oct3/4 (Pou5f1), Sox2, Klf4, and Myc, collectively called OSKM. How fibroblast cells can be remodeled into embryonic stem cell (ESC)-like iPSCs despite high epigenetic barriers has opened a new essential avenue to understanding the action of TFs in developmental regulation. Two forerunning investigations preceded the iPSC phenomenon: exogenous TF-mediated cell remodeling driven by the action of MyoD, and the "pioneer TF" action to preopen chromatin, allowing multiple TFs to access enhancer sequences. The process of remodeling somatic cells into iPSCs has been broken down into multiple subprocesses: the initial attack of OSKM on closed chromatin, sequential changes in cytosine modification, enhancer usage, and gene silencing and activation. Notably, the OSKM TFs change their genomic binding sites extensively. The analyses are still at the descriptive stage, but currently available information is discussed in this chapter.

Estrogen Receptor Chromatin Profiling by CUT&RUN.

Gonadal steroid hormones, namely, testosterone, progesterone, and estrogens, influence the physiological state of an organism through the regulation of gene transcription. Steroid hormones activate nuclear hormone receptor (HR), transcription factors (TFs), which bind DNA in a tissue- and cell type-specific manner to influence cellular function. Identifying the genomic binding sites of HRs is essential to understanding mechanisms of hormone signaling across tissues and disease contexts. Traditionally, chromatin immunoprecipitation followed by sequencing (ChIP-seq) has been used to map the genomic binding of HRs in cancer cell lines and large tissues. However, ChIP-seq lacks the sensitivity to detect TF binding in small numbers of cells, such as genetically defined neuronal subtypes in the brain. Cleavage Under Targets & Release Under Nuclease (CUT&RUN) resolves most of the technical limitations of ChIP-seq, enabling the detection of protein-DNA interactions with as few as 100-1000 cells. In this chapter, we provide a stepwise CUT&RUN protocol for detecting and analyzing the genome-wide binding of estrogen receptor α (ERα) in mouse brain tissue. The steps described here can be used to identify the genomic binding sites of most TFs in the brain.

Mechanisms of PML::Rara and GATA2 Protein Interactions in Hematopoietic Cells

Despite excellent outcomes for Acute Promyelocytic Leukemia (APL) patients with combined ATRA and Arsenic Trioxide therapy, the underlying molecular mechanisms by which the PML::RARA fusion oncoprotein initiates APL are not yet clear. We recently found that the ability of PML::RARA to initiate aberrant serial replating (a surrogate for self-renewal) is dependent upon its ability to bind to DNA, since PML::RARA with a missense mutation (C88A) that prevents the binding of RARA to DNA eliminates aberrant replating. Currently, however, there is no consensus regarding the genomic binding sites of PML::RARA in myeloid progenitor cells and APL cells. Also, the transcriptional and epigenetic consequences of PML::RARA expression in primary hematopoietic cells remain unclear. To define the genomic binding sites of PML::RARA, we generated V5 epitope tagged PML::RARA constructs in MSCV-based retroviral vectors, and transduced mouse hematopoietic stem progenitor cells (HSPCs) with retroviruses containing V5-PML::RARA WT, V5-PML::RARA C88A, or an empty vector. Anti-V5 ChIP-seq revealed that PML::RARA binds to 2,677 distinct loci in primary mouse hematopoietic cells, and 2,064 sites in primary human hematopoietic cells. We previously demonstrated that Gata2 expression is induced with PML::RARA, and indeed, we identified strong PML::RARA binding to the distal Gata2 enhancer in both human and mouse HSPCs. Surprisingly, ATAC-seq revealed that the vast majority of PML::RARA binding sites are found in regions that are chromatin-accessible even in WT promyelocytes, suggesting that these regions are “open” prior to PML::RARA expression, perhaps because of the normal binding of “pioneer” transcription factors to these regions in WT cells. Indeed, evaluation of transcription factor binding motifs in the regions occupied by PML::RARA revealed significant enrichment for RARA binding motifs, and also GATA motifs. We therefore performed ChIP-seq on GATA2 in HSPCs, which confirmed enrichment in binding at the same sites. These data suggested that PML::RARA and GATA2 may bind in close proximity at hundreds of loci in the genome. To test this, we performed proximity labeling studies followed by mass spectrometry using PML::RARA WT vs. PML::RARA C88A fused to the biotin ligase TurboID in primary HSPCs. This approach revealed that the interactions of several proteins with PML::RARA (including GATA2) are dependent on the binding of PML::RARA to DNA; this suggests that DNA may in fact act as a scaffold to stabilize these protein:protein interactions ( Figure 1). To determine whether Gata2 may induce increased chromatin accessibility following PML::RARA expression, we performed ATAC-seq following Gata2 or PML::RARA overexpression in WT hematopoietic progenitor cells. 3,157 regions shared increased accessibility with either PML::RARA or Gata2 overexpression. Moreover, at a transcriptional level, we found that 535 (27.4%) of PML::RARA differentially expressed genes were coordinately regulated by Gata2 in WT cells. This suggests that PML::RARA indirectly regulates many of its gene targets via the upregulation of Gata2, and that Gata2 may be required for PML::RARA to initiate its transcriptional program. To test this, we performed serial replating assays using cells where Gata2 was knocked out either before or after PML::RARA expression. Consistent with our previous study, knock out of Gata2 following PML::RARA expression led to increased self-renewal capacity. However, knock out of Gata2 before PML::RARA expression prevented self-renewal. These data suggest a model in which GATA2 “opens” specific chromatin regions in early myeloid cells, which are then accessible to RARA or PML::RARA. The two proteins interact at these sites, tethered by their mutual interactions to their respective DNA binding motifs. PML::RARA binding promotes a pre-leukemic transcriptional program mediated in part by positively regulating Gata2. Gata2 is therefore initially required for PML::RARA to establish its transcriptional program. However, once that program is established, Gata2 paradoxically acts as a tumor suppressor, performing a normal role to limit proliferative excess in hematopoietic stem and progenitor cells.

Zfp281 and Zfp148 control CD4 + T cell thymic development and T H 2 functions

How CD4 + lineage gene expression is initiated in differentiating thymocytes remains poorly understood. Here, we show that the paralog transcription factors Zfp281 and Zfp148 control both this process and cytokine expression by T helper cell type 2 (T H 2) effector cells. Genetic, single-cell, and spatial transcriptomic analyses showed that these factors promote the intrathymic CD4 + T cell differentiation of class II major histocompatibility complex (MHC II)–restricted thymocytes, including expression of the CD4 + lineage–committing factor Thpok. In peripheral T cells, Zfp281 and Zfp148 promoted chromatin opening at and expression of T H 2 cytokine genes but not of the T H 2 lineage–determining transcription factor Gata3. We found that Zfp281 interacts with Gata3 and is recruited to Gata3 genomic binding sites at loci encoding Thpok and T H 2 cytokines. Thus, Zfp148 and Zfp281 collaborate with Gata3 to promote CD4 + T cell development and T H 2 cell responses.

Genome-wide census of ATF4 binding sites and functional profiling of trait-associated genetic variants overlapping ATF4 binding motifs.

Activating Transcription Factor 4 (ATF4) is an important regulator of gene expression in stress responses and developmental processes in many cell types. Here, we catalogued ATF4 binding sites in the human genome and identified overlaps with trait-associated genetic variants. We probed these genetic variants for allelic regulatory activity using a massively parallel reporter assay (MPRA) in HepG2 hepatoma cells exposed to tunicamycin to induce endoplasmic reticulum stress and ATF4 upregulation. The results revealed that in the majority of cases, the MPRA allelic activity of these SNPs was in agreement with the nucleotide preference seen in the ATF4 binding motif from ChIP-Seq. Luciferase and electrophoretic mobility shift assays in additional cellular models further confirmed ATF4-dependent regulatory effects for the SNPs rs532446 (GADD45A intronic; linked to hematological parameters), rs7011846 (LPL upstream; myocardial infarction), rs2718215 (diastolic blood pressure), rs281758 (psychiatric disorders) and rs6491544 (educational attainment). CRISPR-Cas9 disruption and/or deletion of the regulatory elements harboring rs532446 and rs7011846 led to the downregulation of GADD45A and LPL, respectively. Thus, these SNPs could represent examples of GWAS genetic variants that affect gene expression by altering ATF4-mediated transcriptional activation.

ChIP-Seq analysis reveals PRKACB as a target gene of HOXC13 involved in rabbit hair follicle development

Dermal papilla cells (DPCs) are key regulators of hair follicle (HF) development and growth, which not only regulate HF growth and cycling but play a role in the pathogenesis of hair loss. The transcription factor Homeobox C13 (HOXC13) can modulate the growth and development of HFs. Nevertheless, the specific genes and pathways regulated by HOXC13 in DPCs have yet to be determined. Thus, to gain a better understanding of genomic binding sites involved in HOXC13-regulated HF development, chromatin immunoprecipitation followed by high throughput sequencing (ChIP-Seq) was performed on rabbit DPCs with pcDNA3.1–3 × Flag-HOXC13 overexpression. A complete set of 9670 enrichment peaks was acquired by applying HOXC13-Flag ChIP. Subsequently, the peak sequence was annotated to the rabbit genome, revealing that 6.1 % of the peaks were identified within in the promoter region. Thereafter, five annotated genes were verified using RT-qPCR. The peak-associated genes were mainly enriched in signaling pathways related to HF development, such as MAPK and PI3K-Akt. Furthermore, by using a dual-luciferase reporter assay, we found that HOXC13 can target the protein kinase cAMP‑dependent catalytic β (PRKACB) promoter region (-1596 ∼ -1107 bp) and inhibit its transcription, which was consistent with data obtained from ChIP-seq analysis. Overexpression of PRKACB gene significantly modulated the expression of BCL2, WNT2, LEF1, and SFRP2 genes related to HF development as determined by RT-qPCR (P < 0.01, P < 0.05). The CCK-8 and flow cytometry assays showed that PRKACB significantly inhibited the proliferation of DPCs and promoted apoptosis (P < 0.01). In conclusion, our research revealed that PRKACB has the potential to serve as a novel target gene of HOXC13, contributing to the regulation of the proliferation and apoptosis of DPCs. The process of identifying global target genes can contribute to the understanding of the intricate pathways that HOXC13 regulates in the growth of HFs.

Decitabine disrupts EBV genomic epiallele DNA methylation patterns around CTCF binding sites to increase chromatin accessibility and lytic transcription in gastric cancer.

Epstein-Barr virus (EBV) latency is controlled by epigenetic silencing by DNA methylation [5-methyl cytosine (5mC)], histone modifications, and chromatin looping. However, how they dictate the transcriptional program in EBV-associated gastric cancers remains incompletely understood. EBV-associated gastric cancer displays a 5mC hypermethylated phenotype. A potential treatment for this cancer subtype is the DNA hypomethylating agent, which induces EBV lytic reactivation and targets hypermethylation of the cellular DNA. In this study, we identified a heterogeneous pool of EBV epialleles within two tumor-derived gastric cancer cell lines that are disrupted with a hypomethylating agent. Stochastic DNA methylation patterning at critical regulatory regions may be an underlying mechanism for spontaneous reactivation. Our results highlight the critical role of epigenetic modulation on EBV latency and life cycle, which is maintained through the interaction between 5mC and the host protein CCCTC-binding factor.