Transcription Factor Partners Research Articles

Motif distribution and DNA methylation underlie distinct Cdx2 binding during development and homeostasis

Transcription factors guide tissue development by binding to developmental stage-specific targets and establishing an appropriate enhancer landscape. In turn, DNA and chromatin modifications direct the genomic binding of transcription factors. However, how transcription factors navigate chromatin features to selectively bind a small subset of all the possible genomic target loci remains poorly understood. Here we show that Cdx2—a lineage defining transcription factor that binds distinct targets in developing versus adult intestinal epithelial cells—has a preferential affinity for a non-canonical CpG-containing motif in vivo. A higher frequency of this motif at embryonic Cdx2 targets and methylated state of the CpG during development enables selective Cdx2 binding and activation of developmental enhancers and genes. In adult cells, demethylation at these enhancers prevents ectopic Cdx2 binding, instead directing Cdx2 to its canonical motif without a CpG. This shift in Cdx2 binding facilitates Ctcf and Hnf4 recruitment, establishing super-enhancers during development and homeostatic enhancers in adult cells, respectively. Induced DNA methylation in adult mouse epithelium or cultured cells recruits Cdx2 to developmental targets, promoting corecruitment of partner transcription factors. Thus, Cdx2’s differential CpG motif preferences enable it to navigate distinct DNA methylation profiles, activating genes specific to appropriate developmental stages.

A new pipeline SPICE identifies novel JUN-IKZF1 composite elements.

Transcription factor partners can cooperatively bind to DNA composite elements to augment gene transcription. Here, we report a novel protein-DNA binding screening pipeline, termed Spacing Preference Identification of Composite Elements (SPICE), that can systematically predict protein binding partners and DNA motif spacing preferences. Using SPICE, we successfully identified known composite elements, such as AP1-IRF composite elements (AICEs) and STAT5 tetramers, and also uncovered several novel binding partners, including JUN-IKZF1 composite elements. One such novel interaction was identified at CNS9, an upstream conserved noncoding region in the human IL10 gene, which harbors a non-canonical IKZF1 binding site. We confirmed the cooperative binding of JUN and IKZF1 and showed that the activity of an IL10-luciferase reporter construct in primary B and T cells depended on both this site and the AP1 binding site within this composite element. Overall, our findings reveal an unappreciated global association of IKZF1 and AP1 and establish SPICE as a valuable new pipeline for predicting novel transcription binding complexes.

A new pipeline SPICE identifies novel JUN-IKZF1 composite elements.

Transcription factor partners can cooperatively bind to DNA composite elements to augment gene transcription. Here, we report a novel protein-DNA binding screening pipeline, termed Spacing Preference Identification of Composite Elements (SPICE), that can systematically predict protein binding partners and DNA motif spacing preferences. Using SPICE, we successfully identified known composite elements, such as AP1-IRF composite elements (AICEs) and STAT5 tetramers, and also uncovered several novel binding partners, including JUN-IKZF1 composite elements. One such novel interaction was identified at CNS9, an upstream conserved noncoding region in the human IL10 gene, which harbors a non-canonical IKZF1 binding site. We confirmed cooperative binding of JUN and IKZF1 and showed that the activity of an IL10 -luciferase reporter construct in primary B and T cells depended on both this site and the AP1 binding site within this composite element. Overall, our findings reveal an unappreciated global association of IKZF1 and AP1 and establish SPICE as a valuable new pipeline for predicting novel transcription binding complexes.

Wheat Tae-MIR1118 Constitutes a Functional Module With Calmodulin TaCaM2-1 and MYB Member TaMYB44 to Modulate Plant Low-N Stress Response.

Distinct target genes are modulated by microRNA members and affect various biological processes associated with abiotic stress responses in plants. In this study, we characterized a functional module comprising miRNA/target and a downstream MYB transcription factor partner, Tae-MIR1118/TaCaM2/TaMYB44, in Triticum aestivum to mediate the plant low-nitrogen (N) stress response. Dual luciferase (LUC) assay and expression analysis indicated that TaCaM2 is regulated by Tae-MIR1118 through a posttranscriptional cleavage mechanism. Reporter LUC activity in N. benthamiana leaves co-transformed with effector CaMV35S::Tae-MIR1118 and reporter TaCaM2::LUC was significantly reduced, and the transcripts of Tae-MIR1118 and TaCaM2 in tissues exhibited converse expression patterns under varying N levels. Specifically, the transcripts of Tae-MIR1118 decreased, whereas those of TaCaM2 increased under low-N stress in a temporal-dependent manner. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assays indicated that TaCaM2 interacted with the MYB transcription factor TaMYB44. Transgene analysis revealed the negative roles of Tae-MIR1118 and the positive functions of TaCaM2 and TaMYB44 in regulating plants for low-N stress adaptation by modulating glutamine synthetase activity, N uptake capacity, and root morphology. Yeast one-hybrid, transcriptional activation, and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-PCR) assays indicated that TaMYB44 could bind to the promoters of genes TaGS2.2, TaNRT2.1, and TaPIN4 and induce transcription of these stress-defensive genes. Knockdown of these three genes reduced GS activity, N accumulation, and root growth traits in plants subjected to N starvation. The yield in the wheat variety panel was highly correlated with the transcripts of Tae-MIR1118, TaCaM2, and TaMYB44 in plants cultured under N-deprived field conditions. A major haplotype of Tae-MIR1118, TaMIR1118-Hap1, enhanced the low-N stress tolerance of plants. Our findings indicate that the Tae-MIR1118/TaCaM2/TaMYB44 pathway primarily affects the low-N response of plants by modulating associated physiological processes.

Genome-wide analysis of Smad and Schnurri transcription factors in C. elegans demonstrates widespread interaction and a function in collagen secretion.

Smads and their transcription factor partners mediate the transcriptional responses of target cells to secreted ligands of the Transforming Growth Factor-β (TGF-β) family, including those of the conserved bone morphogenetic protein (BMP) family, yet only a small number of direct target genes have been well characterized. In C. elegans, the BMP2/4 ortholog DBL-1 regulates multiple biological functions, including body size, via a canonical receptor-Smad signaling cascade. Here, we identify functional binding sites for SMA-3/Smad and its transcriptional partner SMA-9/Schnurri based on ChIP-seq peaks (identified by modEncode) and expression differences of nearby genes identified from RNA-seq analysis of corresponding mutants. We found that SMA-3 and SMA-9 have both overlapping and unique target genes. At a genome-wide scale, SMA-3/Smad acts as a transcriptional activator, whereas SMA-9/Schnurri direct targets include both activated and repressed genes. Mutations in sma-9 partially suppress the small body size phenotype of sma-3, suggesting some level of antagonism between these factors and challenging the prevailing model for Schnurri function. Functional analysis of target genes revealed a novel role in body size for genes involved in one-carbon metabolism and in the endoplasmic reticulum (ER) secretory pathway, including the disulfide reductase dpy-11. Our findings indicate that Smads and SMA-9/Schnurri have previously unappreciated complex genetic and genomic regulatory interactions that in turn regulate the secretion of extracellular components like collagen into the cuticle to mediate body size regulation.

Redistribution of PU.1 partner transcription factor RUNX1 binding secures cell survival during leukemogenesis

Transcription factors (TFs) orchestrating lineage-development often control genes required for cellular survival. However, it is not well understood how cells survive when such TFs are lost, for example in cancer. PU.1 is an essential TF for myeloid fate, and mice with downregulated PU.1 levels develop acute myeloid leukemia (AML). Combining a multi-omics approach with a functional genetic screen, we reveal that PU.1-downregulated cells fundamentally change their survival control from cytokine-driven pathways to overexpression of an autophagy-predominated stem cell gene program, for which we also find evidence in human AML. Control of this program involves redirected chromatin occupancy of the PU.1 partner TF Runx1 to a lineage-inappropriate binding site repertoire. Hence, genomic reallocation of TF binding upon loss of a partner TF can act as a pro-oncogenic failsafe mechanism by sustaining cell survival during leukemogenesis.

Functionally redundant roles of ID family proteins in spermatogonial stem cells

Spermatogonial stem cells (SSCs) are essential for sustained sperm production, but SSC regulatory mechanisms and markers remain poorly defined. Studies have suggested that the Id family transcriptional regulator Id4 is expressed in SSCs and involved in SSC maintenance. Here, we used reporter and knockout models to define the expression and function of Id4 in the adult male germline. Within the spermatogonial pool, Id4 reporter expression and inhibitor of DNA-binding 4 (ID4) protein are found throughout the GFRα1+ fraction, comprising the self-renewing population. However, Id4 deletion is tolerated by adult SSCs while revealing roles in meiotic spermatocytes. Cultures of undifferentiated spermatogonia could be established following Id4 deletion. Importantly, ID4 loss in undifferentiated spermatogonia triggers ID3 upregulation, and both ID proteins associate with transcription factor partner TCF3 in wild-type cells. Combined inhibition of IDs in cultured spermatogonia disrupts the stem cell state and blocks proliferation. Our data therefore demonstrate critical but functionally redundant roles of IDs in SSC function.

TEAD1 is crucial for developmental myelination, Remak bundles, and functional regeneration of peripheral nerves.

Previously we showed that the hippo pathway transcriptional effectors, YAP and TAZ, are essential for Schwann cells (SCs) to develop, maintain and regenerate myelin . Although TEAD1 has been implicated as a partner transcription factor, the mechanisms by which it mediates YAP/TAZ regulation of SC myelination are unclear. Here, using conditional and inducible knockout mice, we show that TEAD1 is crucial for SCs to develop and regenerate myelin. It promotes myelination by both positively and negatively regulating SC proliferation, enabling Krox20/Egr2 to upregulate myelin proteins, and upregulating the cholesterol biosynthetic enzymes FDPS and IDI1. We also show stage-dependent redundancy of TEAD1 and that non-myelinating SCs have a unique requirement for TEAD1 to enwrap nociceptive axons in Remak bundles. Our findings establish TEAD1 as a major partner of YAP/TAZ in developmental myelination and functional nerve regeneration and as a novel transcription factor regulating Remak bundle integrity.

Peak Scores Significantly Depend on the Relationships between Contextual Signals in ChIP-Seq Peaks.

Chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) is a central genome-wide method for in vivo analyses of DNA-protein interactions in various cellular conditions. Numerous studies have demonstrated the complex contextual organization of ChIP-seq peak sequences and the presence of binding sites for transcription factors in them. We assessed the dependence of the ChIP-seq peak score on the presence of different contextual signals in the peak sequences by analyzing these sequences from several ChIP-seq experiments using our fully enumerative GPU-based de novo motif discovery method, Argo_CUDA. Analysis revealed sets of significant IUPAC motifs corresponding to the binding sites of the target and partner transcription factors. For these ChIP-seq experiments, multiple regression models were constructed, demonstrating a significant dependence of the peak scores on the presence in the peak sequences of not only highly significant target motifs but also less significant motifs corresponding to the binding sites of the partner transcription factors. A significant correlation was shown between the presence of the target motifs FOXA2 and the partner motifs HNF4G, which found experimental confirmation in the scientific literature, demonstrating the important contribution of the partner transcription factors to the binding of the target transcription factor to DNA and, consequently, their important contribution to the peak score.

Differentiation of Acute Myeloid Leukemia Cells upon Pharmacological Inhibition of LSD1 Requires Its N-Terminal Intrinsically Disordered Region

Lysine-specific histone demethylase 1 (LSD1/KDM1A) governs hematopoiesis by regulating hematopoietic stem cell renewal and proper differentiation. Additionally, LSD1 is required for the maintenance of acute myeloid leukemia (AML) cells and has emerged as a key therapeutic target. Prior work from our group and others have revealed that the LSD1 demethylase activity is not required for AML proliferation. Instead, the interaction between LSD1 and GFI1/GFI1B is necessary to sustain AML cell survival. Thus, beyond its canonical demethylase activity, LSD1 has nonenzymatic, transcription factor (TF)-specific scaffolding functions critical for its function. Most LSD1 inhibitors function by blocking protein-protein interactions between LSD1 and TFs, even though they were originally designed as enzyme inhibitors. While studies indicate that LSD1 has important scaffolding functions, how LSD1 can coordinate with TFs to control gene expression programs is not well understood. Here, we used CRISPR-suppressor scanning to identify regions of LSD1 outside the active site that when mutated can lead to resistance to the covalent LSD1 inhibitor, GSK-LSD1. The understudied N-terminal intrinsically disordered region (IDR) of LSD1 was among the most enriched regions. To explore this further, we generated clonal AML cell lines harboring in-frame deletions within the IDR. These IDR-mutant cell lines were drug resistant and failed to differentiate upon GSK-LSD1 treatment. Moreover, knockdown of endogenous LSD1 followed by overexpression of LSD1 IDR-mutants rescued growth in the presence of GSK-LSD1, confirming that a fully functional IDR is necessary for differentiation. We performed LSD1 co-immunoprecipitation followed by mass spectrometry in wild-type and IDR-mutant cells to measure changes in LSD1 interactors upon GSK-LSD1 treatment. GSK-LSD1 treatment disrupted the LSD1-GFI1B interaction in both wild-type and mutant cells at comparable levels. Since GSK-LSD1 still induces dissociation of the mutant complex but has minimal effect on proliferation, our results suggest that the IDR-mutant cells no longer require GFI1B. In support, knockout of GFI1B inhibited growth of wild-type but not IDR-mutant cells. Strikingly, GSK-LSD1 treatment promoted LSD1's interaction with several master regulator TFs in hematopoiesis, including C/EBPα, PU.1, and RUNX1. Notably, C/EBPα was the most up-regulated interactor in both cell lines, with modest enrichment in IDR-mutant over wild-type. Using live-cell imaging assays, we found that deletions within the LSD1 IDR enhance association with these TFs. These results suggest that GSK-LSD1 reprograms LSD1-TF interactions, disrupting LSD1-GFI1B to induce LSD1-C/EBPα association, and that the IDR mutations alters this LSD1 redistribution to other TF partners. LSD1 is known to silence gene enhancers during differentiation in a process termed “enhancer decommissioning”. The mechanisms by which LSD1 occupies and controls enhancers remain elusive. To test if the LSD1 IDR regulates enhancer elements, we profiled LSD1 and H3K27ac using ChIP-seq in wild-type and IDR-mutant cells upon GSK-LSD1 treatment. Upon GSK-LSD1 treatment, disruption of LSD1-GFI1B binding was accompanied by increased LSD1 levels at other TF and H3K27ac sites, supporting the notion that loss of LSD1-GFI1B interaction induces LSD1 redistribution to other TF partner sites. A concomitant increase in H3K27ac levels was observed across LSD1 sites co-bound by C/EBPα, PU.1, or RUNX1 in wild-type cells. By contrast, in IDR-mutant cells, increases in H3K27ac levels were blocked at these sites upon GSK-LSD1 treatment, suggesting that full enhancer activation is impaired by mutations in the IDR, potentially due to altered association with master regulator TFs. This points to a function of LSD1 IDR in controlling enhancer activation for differentiation. Collectively, our study reveals a role for IDR in regulating LSD1-TF interactions, controlling enhancer activation that is necessary for AML cell differentiation. These data provide deep functional and mechanistic insights into the role of LSD1 IDR and offer important considerations for pharmacological targeting of protein-protein interactions.

FoxK1 associated gene regulatory network in hepatic insulin action and its relationship to FoxO1 and insulin receptor mediated transcriptional regulation

ObjectiveInsulin acts on the liver via changes in gene expression to maintain glucose and lipid homeostasis. This study aimed to the Forkhead box protein K1 (FOXK1) associated gene regulatory network as a transcriptional regulator of hepatic insulin action and to determine its role versus FoxO1 and possible actions of the insulin receptor at the DNA level. MethodsGenome-wide analysis of FoxK1 binding were studied by chromatin immunoprecipitation sequencing and compared to those for IR and FoxO1. These were validated by knockdown experiments and gene expression analysis. ResultsChromatin immunoprecipitation (ChIP) sequencing shows that FoxK1 binds to the proximal promoters and enhancers of over 4000 genes, and insulin enhances this interaction for about 75% of them. These include genes involved in cell cycle, senescence, steroid biosynthesis, autophagy, and metabolic regulation, including glucose metabolism and mitochondrial function and are enriched in a TGTTTAC consensus motif. Some of these genes are also bound by FoxO1. Comparing this FoxK1 ChIP-seq data to that of the insulin receptor (IR) reveals that FoxK1 may act as the transcription factor partner for some of the previously reported roles of IR in gene regulation, including for LARS1 and TIMM22, which are involved in rRNA processing and cell cycle. ConclusionThese data demonstrate that FoxK1 is an important regulator of gene expression in response to insulin in liver and may act in concert with FoxO1 and IR in regulation of genes in metabolism and other important biological pathways.

A Novel Irreversible TEAD Inhibitor, SWTX-143, Blocks Hippo Pathway Transcriptional Output and Causes Tumor Regression in Preclinical Mesothelioma Models.

The Hippo pathway and its downstream effectors, the YAP and TAZ transcriptional coactivators, are deregulated in multiple different types of human cancer and are required for cancer cell phenotypes in vitro and in vivo, while largely dispensable for tissue homeostasis in adult mice. YAP/TAZ and their main partner transcription factors, the TEAD1-4 factors, are therefore promising anticancer targets. Because of frequent YAP/TAZ hyperactivation caused by mutations in the Hippo pathway components NF2 and LATS2, mesothelioma is one of the prime cancer types predicted to be responsive to YAP/TAZ-TEAD inhibitor treatment. Mesothelioma is a devastating disease for which currently no effective treatment options exist. Here, we describe a novel covalent YAP/TAZ-TEAD inhibitor, SWTX-143, that binds to the palmitoylation pocket of all four TEAD isoforms. SWTX-143 caused irreversible and specific inhibition of the transcriptional activity of YAP/TAZ-TEAD in Hippo-mutant tumor cell lines. More importantly, YAP/TAZ-TEAD inhibitor treatment caused strong mesothelioma regression in subcutaneous xenograft models with human cells and in an orthotopic mesothelioma mouse model. Finally, SWTX-143 also selectively impaired the growth of NF2-mutant kidney cancer cell lines, suggesting that the sensitivity of mesothelioma models to these YAP/TAZ-TEAD inhibitors can be extended to other tumor types with aberrations in Hippo signaling. In brief, we describe a novel and specific YAP/TAZ-TEAD inhibitor that has potential to treat multiple Hippo-mutant solid tumor types.

Jasmonates regulate apical hook development by repressing brassinosteroid biosynthesis and signaling.

An apical hook is a special structure formed during skotomorphogenesis in dicotyledonous plant species. It is critical for protecting the shoot apical meristem from mechanical damage during seed germination and hypocotyl elongation in soil. Brassinosteroid (BR) and jasmonate (JA) phytohormones antagonistically regulate apical hook formation. However, the interrelationship between BRs and JAs in this process has not been well elucidated. Here, we reveal that JAs repress BRs to regulate apical hook development in Arabidopsis (Arabidopsis thaliana). Exogenous application of methyl jasmonate (MeJA) repressed the expression of the rate-limiting BR biosynthetic gene DWARF4 (DWF4) in a process relying on 3 key JA-dependent transcription factors, MYC2, MYC3, and MYC4. We demonstrated that MYC2 interacts with the critical BR-activated transcription factor BRASSINAZOLE RESISTANT 1 (BZR1), disrupting the association of BZR1 with its partner transcription factors, such as those of the PHYTOCHROME INTERACTING FACTOR (PIF) family and downregulating the expression of their target genes, such as WAVY ROOT GROWTH 2 (WAG2), encoding a protein kinase essential for apical hook development. Our results indicate that JAs not only repress the expression of BR biosynthetic gene DWF4 but, more importantly, attenuate BR signaling by inhibiting the transcriptional activation of BZR1 by MYC2 during apical hook development.

Reduced YAP1 and FOLR1 in gliomas predict better response to chemotherapeutics

Gliomas harbouring mutations in IDH1 (isocitrate dehydrogenase 1) are characterized by greater sensitivity to chemotherapeutics. These mutants also exhibit diminished levels of transcriptional coactivator YAP1 (yes-associated protein 1). Enhanced DNA damage in IDH1 mutant cells, as evidenced by γH2AX formation (phosphorylation of histone variant H2A.X) and ATM (serine/threonine kinase; ataxia telangiectasia mutated) phosphorylation, was accompanied by reduced FOLR1 (folate receptor 1) expression. Diminished FOLR1, concomitant with heightened γH2AX levels, was also observed in patient-derived IDH1 mutant glioma tissues. Chromatin immunoprecipitation, overexpression of mutant YAP1, and treatment with YAP1-TEAD (TEA domain transcription factors) complex inhibitor verteporfin demonstrated regulation of FOLR1 expression by YAP1 and its partner transcription factor TEAD2. TCGA (The Cancer Genome Atlas) data analysis demonstrated better patient survival with reduced FOLR1 expression. Depletion of FOLR1 rendered IDH1 wild-type gliomas more susceptible to temozolomide-mediated death. Despite heightened DNA damage, IDH1 mutants exhibited reduced levels of IL6 (interleukin 6) and IL8 (interleukin 8) – pro-inflammatory cytokines known to be associated with persistent DNA damage. While both FOLR1 and YAP1 influenced DNA damage, only YAP1 was involved in regulating IL6 and IL8. ESTIMATE and CIBERSORTx analyses revealed the association between YAP1 expression and immune cell infiltration in gliomas. By identifying the influence of YAP1-FOLR1 link in DNA damage, our findings suggest that simultaneous depletion of both could amplify the potency of DNA damaging agents, while concomitantly reducing the release of inflammatory mediators and potentially affecting immune modulation. This study also highlights the novel role of FOLR1 as a probable prognostic marker in gliomas, predicting responsiveness to temozolomide and other DNA damaging agents.

Abstract IA006: Systematic functional screening of chromatin factors identifies strong lineage and disease dependencies in normal and malignant haematopoiesis

Abstract Interactions between transcription factors (TF) and chromatin factors (CF) regulate gene expression programmes to determine cellular fate. However, unlike for TF, the exact role of CF in this process is poorly understood. Using haematopoiesis as a model system, we address this fundamental question in lineage choice of normal haematopoiesis and the maintenance of acute myeloid leukaemia (AML). To do this, we have functionally characterized the requirement ex vivo of 550 CFs in normal lineage differentiation and further dissected, with single-cell resolution, the roles of 40 CFs during in vivo lineage commitment (using Perturb-seq). Further, utilizing epigenetic profiling (ATAC-seq and ChIP-seq) we have probed TF-CF interactions that have lineage regulatory potential. Finally, we compare the functions of key CFs and their TF-associations between normal and malignant haematopoiesis. These studies demonstrate, both ex vivo and in vivo, that similar complexes, including three MLL-H3K4 COMPASS methyltransferases (KMTs) and non-canonical (nc-BAF) and canonical BAF (c-BAF) complexes, regulate disparate lineage decisions across haematopoiesis. However, somewhat paradoxically, we demonstrate that unrelated Repressive complexes function similarly, but non-redundantly, to restrain excessive myeloid differentiation and protect lineage diversity. We identify interactions between CF and TF that explain the regulatory function of CF and demonstrate that KO of the nc-BAF member Brd9 leads to a premalignant accumulation of myeloid progenitors, related to impaired recruitment of late myeloid TFs. Finally, utilizing similar experiments in a relevant murine AML model, we identify perturbations of MLL-, BAF- and Repressive CF that trigger partial differentiation and loss of leukaemia potential. Our data also highlight opposing functions for the same CF in normal and malignant haematopoiesis. CFs such as COMPASS members and cBAF remodelers are critical for the balanced differentiation of specific normal haematopoietic lineages, however the same CF are critical for the maintenance of leukaemia fitness, where they act predominantly to maintain differentiation blockade. Finally, we demonstrate that this altered effect occurs through the differential utilization of TF by CF complexes between normal and malignant haematopoiesis, such as corrupted Stat5a-CF interactions, potentially identifying specific CF-TF complexes as therapeutic targets in AML. Taken all together, our work greatly elucidates the role of CF and their TF partners across normal and malignant haematopoiesis. Citation Format: Brian Huntly. Systematic functional screening of chromatin factors identifies strong lineage and disease dependencies in normal and malignant haematopoiesis. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA006.

Functional Characterization of an In-Frame Deletion in the Basic Domain of the Retinal Transcription Factor ATOH7.

Basic helix–loop–helix (bHLH) transcription factors are evolutionarily conserved and structurally similar proteins important in development. The temporospatial expression of atonal bHLH transcription factor 7 (ATOH7) directs the differentiation of retinal ganglion cells and mutations in the human gene lead to vitreoretinal and/or optic nerve abnormalities. Characterization of pathogenic ATOH7 mutations is needed to understand the functions of the conserved bHLH motif. The published ATOH7 in-frame deletion p.(Arg41_Arg48del) removes eight highly conserved amino acids in the basic domain. We functionally characterized the mutant protein by expressing V5-tagged ATOH7 constructs in human embryonic kidney 293T (HEK293T) cells for subsequent protein analyses, including Western blot, cycloheximide chase assays, Förster resonance energy transfer fluorescence lifetime imaging, enzyme-linked immunosorbent assays and dual-luciferase assays. Our results indicate that the in-frame deletion in the basic domain causes mislocalization of the protein, which can be rescued by a putative dimerization partner transcription factor 3 isoform E47 (E47), suggesting synergistic nuclear import. Furthermore, we observed (i) increased proteasomal degradation of the mutant protein, (ii) reduced protein heterodimerization, (iii) decreased DNA-binding and transcriptional activation of a reporter gene, as well as (iv) inhibited E47 activity. Altogether our observations suggest that the DNA-binding basic domain of ATOH7 has additional roles in regulating the nuclear import, dimerization, and protein stability.

Identification of potential short linear motifs (SLiMs) in intrinsically disordered sequences of proteins by fast time-scale backbone dynamics

Intrinsically disordered regions (IDRs) of proteins contain functionally important short linear motifs (SLiMs), which are composed of 3 to 10 residues. They are often crucial members in protein interaction networks (PINs) in various biological pathways. These short motifs are hard to identify and structurally characterize as very few techniques are amenable for studying IDRs. NMR spectroscopy has emerged as a powerful technique to study IDRs. Here we show that fast picosecond - nanosecond dynamics can be used to identify short rigid segments with significantly lower backbone flexibility compared to the rest of the IDR. Typically, the order parameter (S2) for the backbone amide (NH) bond vector is used to measure residue-wise rigidity. We show that the parameter R1R2/(1-NOE) can be conveniently used to measure residue-wise rigidity for disordered regions. Both the parameters S2 and R1R2/(1-NOE) identify the same rigid segments in the disordered region of the transcription factor SCR. Previously, we have shown that one of the identified rigid segments is indeed a SLiM and specifically interacts with a partner transcription factor. Furthermore, mutations were designed to introduce ionic interactions in a flexible linker connecting two rigid segments. Both the parameters S2 and R1R2/(1-NOE) detect increased rigidity of this flexible linker in the mutants, which was not detected from backbone chemical shifts. Also, no change in global hydrodynamics properties were observed from size exclusion chromatography. Thus, backbone dynamics is highly sensitive to residue-wise rigidity in disordered sequences, which can be conveniently determined from R1R2/(1-NOE) and can identify potential functional SLiMs.

Vitamin D Receptor Binding with DNA in Duodenal Crypt, Duodenal Villi, and Colonic Epithelial Cells of Mice

Vitamin D receptor (VDR) is a transcription factor that mediates calcium absorption by intestinal epithelial cells. Although calcium absorption is ca-nonically thought to occur only in the small intestine, recent studies have shown that VDR activity in the co-lon alone is sufficient to prevent calcium deficiency in mice. Here, we further investigate VDR activity in the colon. We assess VDR-DNA binding in mouse duodenal crypt, duodenal villi, and colonic epithelial cells using Chromatin Immunoprecipitation se-quencing (ChIP-seq). We find that most VDR-respon-sive elements are common to all intestinal epithelial cells, though some VDR-responsive elements are re-gionally-enriched and exhibit greater VDR-binding affinity in either duodenal epithelial cells or colonic epithelial cells. We also assess chromatin accessibil-ity in the same three cell types using Assay for Trans-posase-Accessible Chromatin sequencing (ATAC-seq). By integrating the VDR ChIP-seq and ATAC-seq data, we find that regionally-enriched VDR-re-sponsive elements exhibit greater chromatin acces-sibility in the region of their enrichment. Finally, we assess the transcription factor motifs present in VDR-responsive elements. We find that duodenum- and colon-enriched VDR-responsive elements exhibit different sets of transcription factor motifs other than VDR, suggesting that VDR may act together with dif-ferent partner transcription factors in the two re-gions. Our work is the first investigation of VDR-DNA binding in the colon and provides a basis for further investigations of VDR activity in the colon.

Design and Characterization of a Cell-Penetrating Peptide Derived from the SOX2 Transcription Factor.

SOX2 is an oncogenic transcription factor overexpressed in nearly half of the basal-like triple-negative breast cancers associated with very poor outcomes. Targeting and inhibiting SOX2 is clinically relevant as high SOX2 mRNA levels are positively correlated with decreased overall survival and progression-free survival in patients affected with breast cancer. Given its key role as a master regulator of cell proliferation, SOX2 represents an important scaffold for the engineering of dominant-negative synthetic DNA-binding domains (DBDs) that act by blocking or interfering with the oncogenic activity of the endogenous transcription factor in cancer cells. We have synthesized an interference peptide (iPep) encompassing a truncated 24 amino acid long C-terminus of SOX2 containing a potential SOX-specific nuclear localization sequence, and the determinants of the binding of SOX2 to the DNA and to its transcription factor binding partners. We found that the resulting peptide (SOX2-iPep) possessed intrinsic cell penetration and promising nuclear localization into breast cancer cells, and decreased cellular proliferation of SOX2 overexpressing cell lines. The novel SOX2-iPep was found to exhibit a random coil conformation predominantly in solution. Molecular dynamics simulations were used to characterize the interactions of both the SOX2 transcription factor and the SOX2-iPep with FGF4-enhancer DNA in the presence of the POU domain of the partner transcription factor OCT4. Predictions of the free energy of binding revealed that the iPep largely retained the binding affinity for DNA of parental SOX2. This work will enable the future engineering of novel dominant interference peptides to transport different therapeutic cargo molecules such as anti-cancer drugs into cells.

Meta-analysis of transcriptomic studies of cytokinin-treated rice roots defines a core set of cytokinin response genes.

Cytokinins regulate diverse aspects of plant growth and development, primarily through modulation of gene expression. The cytokinin-responsive transcriptome has been thoroughly described in dicots, especially Arabidopsis, but much less so in monocots. Here, we present a meta-analysis of five different transcriptomic analyses of rice (Oryza sativa) roots treated with cytokinin, including three previously unpublished experiments. We developed a treatment method in which hormone is added to the media of rice seedlings grown in sterile hydroponic culture under a continuous airflow, which resulted in minimal perturbation of the seedlings, thus greatly reducing changes in gene expression in the absence of exogenous hormone. We defined a core set of 205 upregulated and 86 downregulated genes that were differentially expressed in at least three of the transcriptomic datasets. This core set includes genes encoding the type-A response regulators (RRs) and cytokinin oxidases/dehydrogenases, which have been shown to be primary cytokinin response genes. GO analysis revealed that the upregulated genes were enriched for terms related to cytokinin/hormone signaling and metabolism, while the downregulated genes were significantly enriched for genes encoding transporters. Variations of type-B RR binding motifs were significantly enriched in the promoters of the upregulated genes, as were binding sites for other potential partner transcription factors. The promoters of the downregulated genes were generally enriched for distinct cis-acting motifs and did not include the type-B RR binding motif. This analysis provides insight into the molecular mechanisms underlying cytokinin action in a monocot and provides a useful foundation for future studies of this hormone in rice and other cereals.