Overlapping Transcription Factor Research Articles

Generating information-dense promoter sequences with optimal string packing.

Dense arrangements of binding sites within nucleotide sequences can collectively influence downstream transcription rates or initiate biomolecular interactions. For example, natural promoter regions can harbor many overlapping transcription factor binding sites that influence the rate of transcription initiation. Despite the prevalence of overlapping binding sites in nature, rapid design of nucleotide sequences with many overlapping sites remains a challenge. Here, we show that this is an NP-hard problem, coined here as the nucleotide String Packing Problem (SPP). We then introduce a computational technique that efficiently assembles sets of DNA-protein binding sites into dense, contiguous stretches of double-stranded DNA. For the efficient design of nucleotide sequences spanning hundreds of base pairs, we reduce the SPP to an Orienteering Problem with integer distances, and then leverage modern integer linear programming solvers. Our method optimally packs sets of 20-100 binding sites into dense nucleotide arrays of 50-300 base pairs in 0.05-10 seconds. Unlike approximation algorithms or meta-heuristics, our approach finds provably optimal solutions. We demonstrate how our method can generate large sets of diverse sequences suitable for library generation, where the frequency of binding site usage across the returned sequences can be controlled by modulating the objective function. As an example, we then show how adding additional constraints, like the inclusion of sequence elements with fixed positions, allows for the design of bacterial promoters. The nucleotide string packing approach we present can accelerate the design of sequences with complex DNA-protein interactions. When used in combination with synthesis and high-throughput screening, this design strategy could help interrogate how complex binding site arrangements impact either gene expression or biomolecular mechanisms in varied cellular contexts.

A multi-layer functional genomic analysis to understand noncoding genetic variation in lipids

A major challenge of genome-wide association studies (GWASs) is to translate phenotypic associations into biological insights. Here, we integrate a large GWAS on blood lipids involving 1.6 million individuals from five ancestries with a wide array of functional genomic datasets to discover regulatory mechanisms underlying lipid associations. We first prioritize lipid-associated genes with expression quantitative trait locus (eQTL) colocalizations and then add chromatin interaction data to narrow the search for functional genes. Polygenic enrichment analysis across 697 annotations from a host of tissues and cell types confirms the central role of the liver in lipid levels and highlights the selective enrichment of adipose-specific chromatin marks in high-density lipoprotein cholesterol and triglycerides. Overlapping transcription factor (TF) binding sites with lipid-associated loci identifies TFs relevant in lipid biology. In addition, we present an integrative framework to prioritize causal variants at GWAS loci, producing a comprehensive list of candidate causal genes and variants with multiple layers of functional evidence. We highlight two of the prioritized genes, CREBRF and RRBP1, which show convergent evidence across functional datasets supporting their roles in lipid biology.

Whole-genome sequencing of glioblastoma reveals enrichment of non-coding constraint mutations in known and novel genes

BackgroundGlioblastoma (GBM) has one of the worst 5-year survival rates of all cancers. While genomic studies of the disease have been performed, alterations in the non-coding regulatory regions of GBM have largely remained unexplored. We apply whole-genome sequencing (WGS) to identify non-coding mutations, with regulatory potential in GBM, under the hypothesis that regions of evolutionary constraint are likely to be functional, and somatic mutations are likely more damaging than in unconstrained regions.ResultsWe validate our GBM cohort, finding similar copy number aberrations and mutated genes based on coding mutations as previous studies. Performing analysis on non-coding constraint mutations and their position relative to nearby genes, we find a significant enrichment of non-coding constraint mutations in the neighborhood of 78 genes that have previously been implicated in GBM. Among them, SEMA3C and DYNC1I1 show the highest frequencies of alterations, with multiple mutations overlapping transcription factor binding sites. We find that a non-coding constraint mutation in the SEMA3C promoter reduces the DNA binding capacity of the region. We also identify 1776 other genes enriched for non-coding constraint mutations with likely regulatory potential, providing additional candidate GBM genes. The mutations in the top four genes, DLX5, DLX6, FOXA1, and ISL1, are distributed over promoters, UTRs, and multiple transcription factor binding sites.ConclusionsThese results suggest that non-coding constraint mutations could play an essential role in GBM, underscoring the need to connect non-coding genomic variation to biological function and disease pathology.

Single cell transcriptome dynamics from pluripotency to FLK1+ mesoderm.

Hemangiogenic progenitors generating blood and endothelial cells are specified from FLK1-expressing (FLK1+) mesoderm by the transcription factor ETV2. FLK1+ mesoderm also contributes to smooth muscle and cardiomyocytes. However, the developmental process of FLK1+ mesoderm generation and its allocation to various cell fates remain obscure. Recent single cell RNA-sequencing studies of early embryos or in vitro-differentiated human embryonic stem (ES) cells have provided unprecedented information on the spatiotemporal resolution of cells in embryogenesis. These snapshots, however, lack information on continuous dynamic developmental processes. Here, we performed single cell RNA sequencing of in vitro-differentiated mouse ES cells to capture the continuous developmental process leading to hemangiogenesis. We found that hemangiogenic progenitors from ES cells develop through intermediate gastrulation stages, which are gradually specified by 'relay'-like highly overlapping transcription factor modules. Moreover, the transcriptional program of the Flk1+ mesoderm was maintained in the smooth muscle lineage, suggesting that smooth muscle is the default fate of Flk1+ mesoderm. We also identified the SRC kinase contributing to ETV2-mediated activation of the hemangiogenic program. This continuous transcriptome map will facilitate both basic and applied studies of mesoderm development.

Drought-responsive genes expressed predominantly in root tissues are enriched with homotypic cis-regulatory clusters in promoters of major cereal crops

The root appears to be the most relevant organ for breeding drought stress tolerance. However, our knowledge about temporal and spatial regulation of drought-associated genes in the root remains fragmented, especially in crop plants. We performed a meta-analysis of expression divergence of essential drought-inducible genes and analyzed their association with cis-elements in model crops and major cereal crops. Our analysis of 42 selected drought-inducible genes revealed that these are expressed primarily in roots, followed by shoot, leaf, and inflorescence tissues, especially in wheat. Quantitative real-time RT-PCR analysis confirmed higher expression of TaDREB2 and TaAQP7 in roots, correlated with extensive rooting and drought-stress tolerance in wheat. A promoter scan up to 2kb upstream of the translation start site using phylogenetic footprinting revealed 708 transcription factor binding sites, including drought response elements (DREs), auxin response elements (AuxREs), MYCREs/MYBREs, ABAREs, and ERD1 in 19 selected genes. Interestingly, these elements were organized into clusters of overlapping transcription factor binding sites known as homotypic clusters (HCTs), which modulate drought physiology in plants. Taken together, these results revealed the expression preeminence of major drought-inducible genes in the root, suggesting its crucial role in drought adaptation. The occurrence of HCTs in drought-inducible genes highlights the putative evolutionary modifications of crop plants in developing drought adaptation. We propose that these DNA motifs can be used as molecular markers for breeding drought-resilient cultivars, particularly in the cereal crops.

Abstract A46: Targeting transcriptional networks for ovarian cancer therapy.

Abstract High grade serous ovarian cancer remains the deadliest of gynecological cancers. Therefore, a better understanding of the molecular events that promote and maintain high grade serous ovarian cancer is necessary to develop new targeted therapy. It is becoming increasingly clear that aberrantly activated or expressed transcription factors play key roles in tumorigenesis by driving tumorigenic phenotypes through modulation of downstream target genes, some of which are transcription factors themselves developing large transcription factor networks. Thus, we hypothesized that targeting one or more aberrantly expressed transcription factor which represent key nodes in transcription factor networks may provide a new avenue for targeted ovarian cancer therapy. To identify such factors, we analyzed genetic abnormalities in transcription factors in ovarian tumors from The Cancer Genome Atlas (TCGA) and ovarian cancer cell lines from the Cancer Cell Line Encyclopedia, and identified the oncogenic transcriptional modulator BCL6 as being amplified in approximately 25% of ovarian cancer patients and cell lines. We have found that BCL6 is expressed and activated in the majority of ovarian cancer cell lines tested. In addition, we determined that inhibition of BCL6 via siRNA or pharmacologic inhibition reduced the viability of ovarian cancer cell lines and cells from primary ovarian tumors. Moreover, we have utilized a three dimensional model of ovarian cancer to better recapitulate the clinical characteristics of ovarian tumors in vitro. Utilizing this system, we found that BCL6 expression was necessary for the formation of spheroids, which are similar in appearance to the in vivo growth of ovarian cancer. Given this important role of BCL6 in ovarian cancer, we next wanted to determine if BCL6 amplification was associated with other genetic features of high grade serous ovarian cancer. Analysis of ovarian cancer tumors from TCGA determined that BCL6 amplification was significantly associated with Myc amplification. To determine if BCL6 and Myc were part of the same transcription factor network, we utilized available ChIP-seq data to determine if there were overlapping binding regions between these factors. We identified over 200 regions within the genome where BCL6 and Myc binding regions overlapped suggesting that they modulate a subset of the same genes. Analysis of both cell lines and primary ovarian cancer by ChIP confirmed that BCL6 and Myc bind to a subset of overlapping regions. Additionally, a number of genes associated with these binding sites were modulated upon BCL6 and Myc inhibition, demonstrating that these factors are part of an overlapping transcription factor network. Importantly, co-treatment of ovarian cancer cell lines with BCL6 and Myc inhibitors resulted in enhanced loss of cell viability suggesting that combination therapy that targets multiple nodes in a network (such as BCL6 and Myc) may be an effective treatment option. Thus, analyzing the interrelationship among oncogenic transcription factors can shed light on ovarian cancer pathogenesis and also aid in the development of novel treatment strategies for ovarian cancer. Citation Format: Sarah R. Walker, Yixi Zhang, Katarina Bartel, Yasmin Kroll, David A. Frank. Targeting transcriptional networks for ovarian cancer therapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A46.

RVarBase: an updated database for regulatory features of human variants.

We present here the rVarBase database (http://rv.psych.ac.cn), an updated version of the rSNPBase database, to provide reliable and detailed regulatory annotations for known and novel human variants. This update expands the database to include additional types of human variants, such as copy number variations (CNVs) and novel variants, and include additional types of regulatory features. Now rVarBase annotates variants in three dimensions: chromatin states of the surrounding regions, overlapped regulatory elements and variants’ potential target genes. Two new types of regulatory elements (lncRNAs and miRNA target sites) have been introduced to provide additional annotation. Detailed information about variants’ overlapping transcription factor binding sites (TFBSs) (often less than 15 bp) within experimentally supported TF-binding regions (∼150 bp) is provided, along with the binding motifs of matched TF families. Additional types of extended variants and variant-associated phenotypes were also added. In addition to the enrichment in data content, an element-centric search module was added, and the web interface was refined. In summary, rVarBase hosts more types of human variants and includes more types of up-to-date regulatory information to facilitate in-depth functional research and to provide practical clues for experimental design.

VISIONET: intuitive visualisation of overlapping transcription factor networks, with applications in cardiogenic gene discovery.

BackgroundExisting de novo software platforms have largely overlooked a valuable resource, the expertise of the intended biologist users. Typical data representations such as long gene lists, or highly dense and overlapping transcription factor networks often hinder biologists from relating these results to their expertise.ResultsVISIONET, a streamlined visualisation tool built from experimental needs, enables biologists to transform large and dense overlapping transcription factor networks into sparse human-readable graphs via numerically filtering. The VISIONET interface allows users without a computing background to interactively explore and filter their data, and empowers them to apply their specialist knowledge on far more complex and substantial data sets than is currently possible. Applying VISIONET to the Tbx20-Gata4 transcription factor network led to the discovery and validation of Aldh1a2, an essential developmental gene associated with various important cardiac disorders, as a healthy adult cardiac fibroblast gene co-regulated by cardiogenic transcription factors Gata4 and Tbx20.ConclusionsWe demonstrate with experimental validations the utility of VISIONET for expertise-driven gene discovery that opens new experimental directions that would not otherwise have been identified.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0578-0) contains supplementary material, which is available to authorized users.

Effect of Focal Ischemia on Long Noncoding RNAs

Long noncoding RNAs (lncRNAs) play a significant role in cellular physiology. We evaluated the effect of focal ischemia on the expression of 8314 lncRNAs in rat cerebral cortex using microarrays. Ischemia was induced by transient middle cerebral artery occlusion. Genomic and transcriptomic correlates of the stroke-responsive lncRNAs and the transcription factor binding sties in their promoters were evaluated with bioinformatics. Three hundred fifty-nine lncRNAs were upregulated (>2-fold) and 84 were downregulated (<0.5-fold) at 3 hours to 12 hours of reperfusion after middle cerebral artery occlusion compared with sham. Sixty-two stroke-responsive lncRNAs showed >90% sequence homology with exons of protein-coding genes. Promoters of stroke-responsive lncRNA genes and their homologous protein-coding genes showed highly overlapping transcription factor binding sites. Despite presence of open reading frames, lncRNAs did not form any product when subjected to in vitro translation. Stroke significantly alters cerebral lncRNA expression profiles.

Nuclear Factor κB- and Specificity Protein 1-dependent p53-mediated Bi-directional Regulation of the Human Manganese Superoxide Dismutase Gene

Tumor suppressor p53 is known to activate certain sets of genes while suppressing others. However, whether p53 can both activate and suppress the same gene is unclear. To address this question, concentration-dependent p53 effect on the manganese superoxide dismutase (MnSOD) gene was investigated. By transfecting p53 in PC-3 cells, we demonstrate that low concentrations of p53 increase while high concentrations suppress MnSOD expression. The physiological relevance of this effect was determined in vitro and in vivo using combined UVB-mediated activation and small interference RNA-mediated suppression of p53. Results were consistent with the bi-directional effect of p53 on MnSOD expression. MnSOD-promoter/enhancer analysis demonstrates that p53 is suppressive to the promoter activity regardless of the presence or absence of putative p53 binding sites. However, a low level of p53 increases MnSOD gene transcription in the presence of the intronic-enhancer element, and this effect is dependent on nuclear-factor kappaB (NF-kappaB) binding sites. Expression of p53 enhances nuclear levels of p65 with corresponding increase in the DNA-binding activity of NF-kappaB as detected by electrophoretic mobility shift and chromatin immunoprecipitation assays. Transfection of p65 small interference RNA reduces the positive effect of p53 on MnSOD gene transcription. These data suggest that p65 can overcome the negative effect of p53 on MnSOD expression. However, when the level of p53 was further increased, the suppressive effect of p53 outweighed the positive effect of p65 and led to the suppression of MnSOD gene transcription. These results demonstrated that p53 can both suppress and induce MnSOD expression depending on the balance of promoter and enhancer binding transcription factors.

Molecular genetic analysis of a human insulin‐like growth factor 1 promoter P1 variation

Insulin-like growth factor 1 (IGF1) exerts important endocrine and paracrine functions in the cardiovascular system. We identified the common variant -1411C>T in the IGF1 upstream promoter P1, located within several overlapping transcription factor binding sites. Using transient transfection assays, we identified this site as a functional enhancer. The T allele-carrying enhancer, compared with the C allelic portion, exerts significantly reduced or even abrogated activity, respectively, in SaOs-2 and HepG2 (all P<0.0001) as well as in differentiated THP-1 macrophages. Electrophoretic mobility shift assay and subsequent supershift experiments in HepG2 identified c-Jun as the binding partner exclusively to the T allele, whereas CCAAT/enhancer-binding protein delta and interferon consensus site-binding protein/interferon-regulating factor 8 interacted only with the C allelic promoter portion. Furthermore, genotyping in a case-control study for essential hypertension (n=745 hypertensive patients; n=769 normotensive control subjects) for this variant revealed an odds ratio for hypertension of 0.73 (95% confidence interval 0.58-0.91, P=0.006) associated with the T allele, and normotensive subjects carrying the protective T allele displayed a significant decrease in diastolic (P=0.036) and systolic (P=0.024) blood pressure levels. We here report detection of a functional enhancer module in the upstream IGF1 promoter region, which might play a key role in local IGF1 bioavailability. Whether -1411C>T is also associated with other IGF1-related disease phenotypes should be evaluated further in population studies.

Transcriptional Dysregulation in Down Syndrome: Predictions for Altered Protein Complex Stoichiometries and Post-translational Modifications, and Consequences for Learning/Behavior Genes ELK, CREB, and the Estrogen and Glucocorticoid Receptors

The phenotype of Down syndrome, trisomy of chromosome 21, is hypothesized to be produced by the increased expression due to gene dosage of normal chromosome 21 genes. Chromosome 21 encodes a number of proteins that, based on experimental evidence or domain composition, are classed as transcription factors or their co-regulators. Other chromosome 21 proteins contribute to post-translational modification of transcription factors, including their phosphorylation, dephosphorylation and sumoylation. Several of these chromosome 21 proteins and the pathways in which they function have overlapping transcription factor specificities. Thus, altered stoichiometry in complexes and altered levels of activation of individual transcription factors may contribute to the Down syndrome phenotype by perturbation of downstream gene expression. Here we review recent data on four chromosome 21 proteins: NRIP1, GABPA, DYRK1A and SUMO3. We discuss the implications for activation of ELK, CREB, C/EBP alpha, beta estrogen and glucocorticoid receptors, and for expression of BDNF. Each of these proteins is relevant to learning, behavior and/or development and therefore perturbation of their activation may contribute to the Down syndrome phenotype.

Transcriptional regulation by competing transcription factor modules.

Gene regulatory networks lie at the heart of cellular computation. In these networks, intracellular and extracellular signals are integrated by transcription factors, which control the expression of transcription units by binding to cis-regulatory regions on the DNA. The designs of both eukaryotic and prokaryotic cis-regulatory regions are usually highly complex. They frequently consist of both repetitive and overlapping transcription factor binding sites. To unravel the design principles of these promoter architectures, we have designed in silico prokaryotic transcriptional logic gates with predefined input–output relations using an evolutionary algorithm. The resulting cis-regulatory designs are often composed of modules that consist of tandem arrays of binding sites to which the transcription factors bind cooperatively. Moreover, these modules often overlap with each other, leading to competition between them. Our analysis thus identifies a new signal integration motif that is based upon the interplay between intramodular cooperativity and intermodular competition. We show that this signal integration mechanism drastically enhances the capacity of cis-regulatory domains to integrate signals. Our results provide a possible explanation for the complexity of promoter architectures and could be used for the rational design of synthetic gene circuits.

The Same Xenobiotic Response Element Is Required for Constitutive and Inducible Expression of the Mammalian Aldehyde Dehydrogenase-3 Gene

The mammalian aldehyde dehydrogenase-3 gene (ALDH3) exhibits several aspects of tissue-specific expression. Certain normal tissues, such as the cornea, constitutively expressALDH3at very high levels. Other tissues, such as normal liver, do not expressALDH3.In liver,ALDH3is inducible by polycyclic aromatic hydrocarbon xenobiotics by an Ah-receptor (AhR)-mediated pathway in which a liganded AhR complexes with nuclear ARNT protein, and the complex binds to a xenobiotic response element (XRE) sequence located near −3.0 kb in theALDH35′ flanking region and initiates transcription. We used our recently developed rat corneal epithelium culture model (Boeschet al., J. Biol. Chem.271, 5150–5157, 1996) to study the molecular basis of constitutiveALDH3expression. Transient transfection assays of corneal epithelium using a battery ofALDH35′ flanking region–CAT reporter gene constructs indicate that high constitutiveALDH3expression involves the samecis-acting elements as xenobiotic-inducedALDH3expression in liver. These elements include a strong basal promoter region and the XRE located near −3.0 kb. Western analysis confirms the presence of AhR and ARNT proteins in 3-methylcholanthrene-treated rat liver, as well as ARNT protein in rat corneal epithelium. No AhR protein is found in rat cornea. The −3.0-kbALDH3XRE region contains multiple overlapping transcription factor binding sequences, including consensus sites for AhR, ARNT, HNF1, HNF4, and C/ebp. Electrophoretic mobility shift assays (EMSAs) indicate that constitutive expression ofALDH3in cornea involves binding of ARNT, HNF1, and HNF4 to theALDH3–XRE in an Ah-receptor-independent, ARNT-requiring manner. Transient transfection ofALDH3–CAT reporter gene constructs possessing a mutation in either the ARNT- or HNF4–DNA binding sites of the XRE confirms the functional importance of these sequence motifs in constitutiveALDH3expression.

NF-κB binding is elevated in hepatoma variant cells lacking the HNF4→HNF1 hierarchical pathway: [formula omitted], Pediatric Research Institute, St. Louis University Health Sciences Center, St. Louis, MO

The mammalian aldehyde dehydrogenase-3 gene (ALDH3) exhibits several aspects of tissue-specific expression. Certain normal tissues, such as the cornea, constitutively expressALDH3at very high levels. Other tissues, such as normal liver, do not expressALDH3.In liver,ALDH3is inducible by polycyclic aromatic hydrocarbon xenobiotics by an Ah-receptor (AhR)-mediated pathway in which a liganded AhR complexes with nuclear ARNT protein, and the complex binds to a xenobiotic response element (XRE) sequence located near −3.0 kb in theALDH35′ flanking region and initiates transcription. We used our recently developed rat corneal epithelium culture model (Boeschet al., J. Biol. Chem.271, 5150–5157, 1996) to study the molecular basis of constitutiveALDH3expression. Transient transfection assays of corneal epithelium using a battery ofALDH35′ flanking region–CAT reporter gene constructs indicate that high constitutiveALDH3expression involves the samecis-acting elements as xenobiotic-inducedALDH3expression in liver. These elements include a strong basal promoter region and the XRE located near −3.0 kb. Western analysis confirms the presence of AhR and ARNT proteins in 3-methylcholanthrene-treated rat liver, as well as ARNT protein in rat corneal epithelium. No AhR protein is found in rat cornea. The −3.0-kbALDH3XRE region contains multiple overlapping transcription factor binding sequences, including consensus sites for AhR, ARNT, HNF1, HNF4, and C/ebp. Electrophoretic mobility shift assays (EMSAs) indicate that constitutive expression ofALDH3in cornea involves binding of ARNT, HNF1, and HNF4 to theALDH3–XRE in an Ah-receptor-independent, ARNT-requiring manner. Transient transfection ofALDH3–CAT reporter gene constructs possessing a mutation in either the ARNT- or HNF4–DNA binding sites of the XRE confirms the functional importance of these sequence motifs in constitutiveALDH3expression.

Hepatocyte nuclear factor 3 determines the amplitude of the glucocorticoid response of the rat tyrosine aminotransferase gene.

Hepatocyte nuclear factor 3 (HNF3) recognizes two apparently distinct classes of sequence. However, a detailed mutational analysis of a representative binding site of each class reveals that these sequences display common features. We propose a unified consensus sequence for HNF3-binding sites. The basis of the sequence specificity of the interaction of HNF3 with DNA is analyzed in light of the recently determined structure of an HNF3-DNA complex (Clark et al., Nature 364, 412-420, 1993). Particularly, our study reveals that the DNA site used for this structural analysis is too short to account for all HNF3-DNA interactions. The better knowledge of the sequence determinant recognized by HNF3 has allowed us to analyze its function in the glucocorticoid response of the rat tyrosine aminotransferase (TAT) gene. This response is mediated through a complex array of neighboring and overlapping transcription factor binding sites. Selective inactivation of the HNF3-binding sites in this glucocorticoid response unit (GRU) allows us to demonstrate unambiguously that they play a major role in the amplitude of the glucocorticoid response. Furthermore, HNF3 beta overexpression results in a stimulation of the glucocorticoid response that is dependent on the integrity of its binding sites. We also show that the relative level of HNF3 determines the extent of the contribution of one of the glucocorticoid receptor binding sites. Our results indicate that HNF3 accounts for most of the liver-specific activity of this GRU.

Transcriptional control of vitamin D-regulated proteins.

Vitamin D is a physiological regulator of gene transcription associated with control of a broad spectrum of biological processes that include but is not restricted to growth, differentiation and calcium-mediated homeostatic control. Transcriptional regulation is mediated by sequence-specific interactions of a 1,25(OH)2D3-vitamin D receptor-accessory factor complex with vitamin D responsive elements (VDRE) residing in the promoters of hormone responsive genes. Functioning primarily as a transcription enhancer, activity at the VDRE is controlled by diverse and integrated cellular signalling pathways acting synergistically and/or antagonistically with a series of basal regulatory elements and other hormone regulated sequences that are components of modularly organized vitamin D-responsive gene promoters. Molecular mechanisms that integrate the activities at promoter elements contributing to vitamin D-related transcriptional control include overlapping transcription factor binding domains within regulatory elements and cooperative activities at independent regulatory sequences that determine the level of vitamin D responsiveness.