Assembly Of Transcription Factor Complexes Research Articles

Mechanism of allele specific assembly and disruption of master regulator transcription factor complexes of NF-KBp50, NF-KBp65 and HIF1a on a non-coding FAS SNP

A challenging question in genetics is to understand the molecular function of non-coding variants of the genome. By using differential EMSA, ChIP and functional genome analysis, we have found that changes in transcription factors (TF) apparent binding affinity and dissociation rates are responsible for allele specific assembly or disruption of master TFs: we observed that NF-KBp50, NF-KBp65 and HIF1a bind with an affinity of up to 10 fold better to the C-allele than to the T-allele of rs7901656 both in vivo and in vitro. Furthermore, we showed that NF-KBp50, p65 and HIF1a form higher order heteromultimeric complexes overlapping rs7901656, implying synergism of action among TFs governing cellular response to infection and hypoxia. With rs7901656 on the FAS gene as a paradigm, we show how allele specific transcription factor complex assembly and disruption by a causal variant contributes to disease and phenotypic diversity. This finding provides the highly needed mechanistic insight into how the molecular etiology of regulatory SNPs can be understood in functional terms.

Abstract 1704: High mobility group A1 chromatin remodeling protein expands the intestinal stem cell compartment and Paneth cell niche through Wnt/β-catenin signaling and Sox9

Abstract The High Mobility Group A1 (HMGA1) gene is overexpressed in most poorly differentiated cancers and high levels portend adverse clinical outcomes, although the molecular mechanisms through which it functions are poorly understood. HMGA1 encodes the HMGA1a and HMGA1b chromatin remodeling proteins, which modulate gene expression by bending chromatin and orchestrating the assembly of transcription factor complexes to DNA. HMGA1 is highly expressed during embryogenesis, but silenced in adult, differentiated tissues. Postnatally, HMGA1 expression is maintained in adult stem cells, such as intestinal stem cells (ISCs); however, its role in this setting has been unknown. Here, we report that Hmga1 overexpression in ISCs of transgenic mice drives expansion in the ISC compartment leading to hyperproliferation, aberrant crypt formation, and polyposis. Surprisingly, Hmga1 transgenic mice also exhibit marked expansion in terminally differentiated Paneth cells, which comprise an epithelial cell niche for ISCs. To dissect the mechanisms mediating these phenotypes, we generated three-dimensional (3D) intestinal organoids with varied expression of Hmga1. Strikingly, silencing Hmga1 in wildtype crypt cells disrupts their ability to organize into functional 3D organoids with bud formation, while crypt cells expressing ectopic Hmga1 exhibit enhanced organoid formation with increased ISC number, proliferation, and bud development. Because Wnt/β-catenin signaling is central to ISC function, we determined whether Hmga1 modulates this pathway. β-catenin protein is increased in the crypts of the Hmga1 transgenic mice and organoids. Hmga1 amplifies Wnt/β-catenin signaling by inducing both genes that encode Wnt cell surface receptors and target genes downstream of Wnt/β-catenin. Hmga1 also directly up-regulates Sox9, which is required for terminal differentiation to Paneth cells. This is the first example of Hmga1 fostering terminal differentiation to establish a stem cell niche. In human intestinal epithelium, HMGA1 and SOX9 are highly correlated (P = 0.008), and both become up-regulated in carcinogenesis. These results reveal a novel role for Hmga1 in intestinal homeostasis by maintaining both the stem cell pool and epithelial niche compartment and suggest that deregulated Hmga1 perturbs this equilibrium during intestinal carcinogenesis. Citation Format: Lingling Xian, Tait Huso, Amy Belton, David Huso, Linda M. S. Resar. High mobility group A1 chromatin remodeling protein expands the intestinal stem cell compartment and Paneth cell niche through Wnt/β-catenin signaling and Sox9. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1704.

Cellular Levels of Signaling Factors Are Sensed by β-actin Alleles to Modulate Transcriptional Pulse Intensity.

The transcriptional response of β-actin to extra-cellular stimuli is a paradigm for transcription factor complex assembly and regulation. Serum induction leads to a precisely timed pulse of β-actin transcription in the cell population. Actin protein is proposed to be involved in this response, but it is not known whether cellular actin levels affect nuclear β-actin transcription. We perturbed the levels of key signaling factors and examined the effect on the induced transcriptional pulse by following endogenous β-actin alleles in single living cells. Lowering serum response factor (SRF) protein levels leads to loss of pulse integrity, whereas reducing actin protein levels reveals positive feedback regulation, resulting in elevated gene activation and a prolonged transcriptional response. Thus, transcriptional pulse fidelity requires regulated amounts of signaling proteins, and perturbations in factor levels eliminate the physiological response, resulting in either tuning down or exaggeration of the transcriptional pulse.

CBFB–MYH11/RUNX1 together with a compendium of hematopoietic regulators, chromatin modifiers and basal transcription factors occupies self-renewal genes in inv(16) acute myeloid leukemia

Different mechanisms for CBFβ-MYH11 function in acute myeloid leukemia with inv(16) have been proposed such as tethering of RUNX1 outside the nucleus, interference with transcription factor complex assembly and recruitment of histone deacetylases, all resulting in transcriptional repression of RUNX1 target genes. Here, through genome-wide CBFβ-MYH11-binding site analysis and quantitative interaction proteomics, we found that CBFβ-MYH11 localizes to RUNX1 occupied promoters, where it interacts with TAL1, FLI1 and TBP-associated factors (TAFs) in the context of the hematopoietic transcription factors ERG, GATA2 and PU.1/SPI1 and the coregulators EP300 and HDAC1. Transcriptional analysis revealed that upon fusion protein knockdown, a small subset of the CBFβ-MYH11 target genes show increased expression, confirming a role in transcriptional repression. However, the majority of CBFβ-MYH11 target genes, including genes implicated in hematopoietic stem cell self-renewal such as ID1, LMO1 and JAG1, are actively transcribed and repressed upon fusion protein knockdown. Together these results suggest an essential role for CBFβ-MYH11 in regulating the expression of genes involved in maintaining a stem cell phenotype.

The hematopoietic master regulator RUNX1 reshapes the epigenetic landscape at the onset of hematopoiesis

Hematopoiesis in the embryo originates from mesodermal cells and proceeds via a common precursor (hemangioblast) of endothelial cells and blood cells. Hemangioblasts give rise to specialised endothelial cells (hemogenic endothelium) which subsequently undergo a transition into hematopoietic precursor cells. These cell fate decisions are governed by lineage-specific transcription factors, such as RUNX1, SCL/TAL1, FLI-1, PU.1 and C/EBP family members. In our work we study how dynamic shifts in the transcriptional regulatory network during this developmental pathway are regulated and how transcription factors control the activation of hematopoietic genes. We are also investigating, how these factors interact with each other and with the chromatin landscape. To this end, we measured the genome-wide dynamics of chromatin alterations during the different steps of formation of the hematopoietic system from mesodermal cells using ES cell differentiation as model. We show that the hematopoietic program is already primed in the hemogenic endothelium as indicated by the binding of SCL/TAL1, FLI-1, C/EBPβ and enhancer-bound RNA-Polymerase II as well as the appearance of DNAsel hypersensitive sites. RUNX1 is absolutely required for the transition from hemogenic endothelium cells into hematopoietic progenitors. To obtain mechanistic information how this factor drives this process, we examined the assembly of hematopoietic transcription factors on their targets before and after this transition. Using an inducible system, we show that after induction RUNX1 binds to primed, but also novel elements and increases their histone acetylation. Moreover, RUNX1 initiates rapid global alterations in the binding patterns of SCL/TAL1 and FLI1, involving both the extinction of binding sites as well as the establishment of new sites. A significant fraction of new elements bind SCL/TAL1 and FLI1 in close proximity to RUNX1 in a pattern that is specific for hematopoietic cells. RUNX1 has previously shown to be expendable in hematopoietic precursor cells once they have formed from the hemogenic endothelium. By precisely timed withdrawal studies we show that immediately after RUNX1 induction altered transcription factor complex assembly at many genes is still reversible, but not at all of them. Our experiments suggest a dynamic interplay between RUNX1 and other transcription factors that dictates the half-life of factor assemblies and their dependency on RUNX1 and give a fascinating insight into how a single master regulator shapes the epigenetic landscape.

HMGA1, a Factor Enriched in Hematopoietic Stem Cells, Embryonic Stem Cells, and Hematologic Malignancy, Enhances Cellular Reprogramming to a Pluripotent Stem-Like Cell.

AbstractAbstract 2323Although recent studies have identified genes important in hematopoietic stem cells (HSCs), human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPSCs), the molecular underpinnings of normal stem cell function are unclear. A better understanding of key stem cell pathways will be essential for the safe use of stem cells in regenerative medicine and should also uncover novel therapeutic targets in aggressive hematologic malignancies and other stem-like cancer cells. To elucidate the molecular underpinnings of “stemness”, we investigated transcriptional networks in pluripotent stem cells. Our focus is the high mobility group A1 (HMGA1) gene, which encodes the HMGA1a and HMGA1b chromatin remodeling proteins. These proteins bind to AT-rich regions of DNA and orchestrate the assembly of transcription factor complexes to alter chromatin structure and modulate gene expression. HMGA1 is highly expressed during embryogenesis with low or undetectable levels in adult, differentiated tissues. HMGA1 is also enriched in HSCs, hESCs, iPSCs, refractory leukemia, and poorly differentiated solid tumors. Our group discovered that HMGA1 functions as a potent oncogene in cultured cells and causes aggressive leukemia in transgenic mice. We also found that high levels of HMGA1 expression correlate with relapse in acute lymphoblastic leukemia. Together, these findings suggest that HMGA1 drives a stem cell phenotype during normal development, hematopoiesis, and malignant transformation.To further investigate the role of HMGA1 in a stem cell state, we compared its expression in iPSCs, hESCs, HSCs, and cancer cells. HMGA1 is highly expressed in fully reprogrammed iPSCs and hESCs, with intermediate levels in HSCs and cancer cells, and low levels in fibroblasts. When hESCs are induced to differentiate, HMGA1 decreases and parallels that of other pluripotency factors. Conversely, forced expression of HMGA1 blocks differentiation in hESCs. We also discovered that HMGA1 enhances cellular reprogramming of somatic cells (mesenchymal stem cells, HSCs, and fetal lung fibroblasts) to an iPSC together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC or OSKM). HMGA1 results in an increase in the number and size of iPSC colonies compared to OSKM controls. Surprisingly, there was normal differentiation in vitro and benign, teratoma formation in vivo of the HMGA1-derived iPSCs. During the reprogramming process, HMGA1 induces the expression of pluripotency genes, including SOX2, LIN28, and cMYC, while knockdown of HMGA1 in hESCs results in the repression of these genes. Chromatin immunoprecipitation shows that HMGA1 binds to the promoters of these pluripotency genes in vivo.In summary, our findings uncover a key role for HMGA1 as a regulator of the stem cell state through transcriptional networks that induce pluripotency and an undifferentiated state. Further studies are needed to determine if HMGA1 pathways could be targeted in hematologic and other malignancies or exploited in regenerative medicine. Disclosures:No relevant conflicts of interest to declare.

Abstract 429: The high mobility group A1 oncogene enhances cellular reprogramming to a pluripotent stem-like cell

Abstract The molecular mechanisms that enable cancer cells to metastasize are poorly understood, although emerging evidence indicates that transcriptional networks required for stem cell properties during embryogenesis are co-opted by cancer cells during metastatic progression. To elucidate the molecular underpinnings of “stemness” in cancer and normal development, we investigated transcriptional networks and epigenetic alterations during the induction of pluripotent stem cells. Our focus is the high mobility group A1 (HMGA1) gene, which encodes proteins that bind to AT-rich regions of DNA and orchestrate the assembly of transcription factor complexes to alter chromatin structure and modulate gene expression. Our group first discovered that HMGA1 functions as a potent oncogene in cultured cells and causes aggressive tumors in transgenic mice. Recent studies also identified HMGA1 as a key transcription factor enriched in human embryonic stem (hESCs) cells, induced pluripotent stem cells (iPSCs), refractory leukemia, and high-grade/poorly differentiated cancers arising from diverse tissues. Together, these findings are consistent with the hypothesis that HMGA1 drives an undifferentiated, stem cell-like state during malignant transformation and normal development. To further investigate the role of HMGA1 in the stem cell state, we assessed its function in the derivation of iPSCs using multiple approaches. Here, we demonstrate for the first time that HMGA1 significantly enhances the reprogramming of somatic cells (bone marrow-derived mesenchymal stem cells, fetal lung cells, or mononuclear blood cells) into iPSCs together with OCT4, SOX2, KLF4, and cMYC (OSKM) using a retroviral or episomal, non-integrating approach. When hESCs are induced to differentiate, HMGA1 expression falls and parallels that of other pluripotency factors, such as OCT4, NANOG, and SOX2. We also found that forced expression of HMGA1 blocks differentiation of hESCs. To determine how HMGA1 induces a stem cell state, we assessed gene expression and epigenetic changes. During the reprogramming process, HMGA1 induces the expression of pluripotency and cancer genes, including SOX2, LIN28, and cMYC, while in hESCs, knock-down of HMGA1 results in repression of these genes. In addition, NANOG and OCT4 are repressed in hESCs following knock-down of HMGA1. By chromatin immunoprecipitation, HMGA1 binds to the promoters of SOX2, LIN28, and cMYC in vivo in hESCs. Moreover, HMGA1 is associated with decreased promoter methylation of select stem cell genes early in reprogramming. These findings uncover a key role for HMGA1 as a regulator of the stem cell state through transcriptional networks and epigenetic remodeling that induce pluripotency and an undifferentiated state. Further studies are needed to determine if HMGA1 pathways could be targeted in poorly differentiated, stem-like cancers or exploited in regenerative medicine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 429. doi:1538-7445.AM2012-429

Protein Stability and Transcription Factor Complex Assembly Determined by the SCL-LMO2 Interaction

Gene expression programs are established by networks of interacting transcription factors. The basic helix-loop-helix factor SCL and the LIM-only protein LMO2 are components of transcription factor complexes that are essential for hematopoiesis. Here we show that LMO2 and SCL are predominant interaction partners in hematopoietic cells and that this interaction occurs through a conserved interface residing in the loop and helix 2 of SCL. This interaction nucleates the assembly of SCL complexes on DNA and is required for target gene induction and for the stimulation of erythroid and megakaryocytic differentiation. We also demonstrate that SCL determines LMO2 protein levels in hematopoietic cells and reveal that interaction with SCL prevents LMO2 degradation by the proteasome. We propose that the SCL-LMO2 interaction couples protein stabilization with higher order protein complex assembly, thus providing a powerful means of modulating the stoichiometry and spatiotemporal activity of SCL complexes. This interaction likely provides a rate-limiting step in the transcriptional control of hematopoiesis and leukemia, and similar mechanisms may operate to control the assembly of diverse protein modules.

An HMG Protein, Hmo1, Associates with Promoters of Many Ribosomal Protein Genes and throughout the rRNA Gene Locus in Saccharomyces cerevisiae

HMG proteins are architectural proteins that bind to DNA with low sequence specificity, but little is known about their genomic location and biological functions. Saccharomyces cerevisiae encodes 10 HMG proteins, including Hmo1, which is important for maximal transcription of rRNA. Here we use chromatin immunoprecipitation coupled with microarray analysis to determine the genome-wide association of Hmo1. Unexpectedly, Hmo1 binds strongly to the promoters of most ribosomal protein (RP) genes and to a number of other specific genomic locations. Hmo1 binding to RP promoters requires Rap1 and (to a lesser extent) Fhl1, proteins that also associate with RP promoters. Hmo1, like Fhl1 and Ifh1, typically associates with an IFHL motif in RP promoters, but deletion of the IFHL motif has a very modest effect on Hmo1 binding. Surprisingly, loss of Hmo1 abolishes binding of Fhl1 and Ifh1 to RP promoters but does not significantly affect the level of transcriptional activity. These results suggest that Hmo1 is required for the assembly of transcription factor complexes containing Fhl1 and Ifh1 at RP promoters and that proteins other than Fhl1 and Ifh1 also play an important role in RP transcription. Lastly, like mammalian UBF, Hmo1 associates at many locations throughout the rRNA gene locus, and it is important for processing of rRNA in addition to its role in rRNA transcription. We speculate that Hmo1 has a role in coordinating the transcription of rRNA and RP genes.

Histone Dynamics on the Interleukin-2 Gene in Response to T-Cell Activation

Several models have been proposed for the mechanism of chromatin remodelling across the promoters of inducible genes in mammalian cells. The most commonly held model is one of cooccupation where histone proteins are modified by acetylation or phosphorylation and nucleosomes are remodelled, allowing the assembly of transcription factor complexes. Using chromatin immunoprecipitation, we observed an apparent decrease of histone acetylation and phosphorylation signals at the proximal promoter region of the inducible interleukin-2 and granulocyte-macrophage colony-stimulating factor genes in response to T-cell activation. We showed that this apparent decrease was due to a loss of histone H3 and H4 proteins corresponding to a decrease in nucleosome occupation of the promoter. This histone loss is reversible; it is dependent on the continual presence of appropriate activating signals and transcription factors and is not dependent on the acetylation status of the histone proteins. These data show for the first time that histone proteins are lost from a mammalian promoter upon activation of transcription and support a model of activation-dependent disassembly and reassembly of nucleosomes.

The mechanism of complex formation between Fli-1 and SRF transcription factors.

The mechanisms of multicomponent transcription factor complex assembly are currently poorly defined. A paradigm for this type of complex is the ETS-domain transcription factor Elk-1 and the MADS-box transcription factor SRF which form a ternary complex with the c- fos serum response element (SRE). In this study we have analysed how a different ETS-domain transcription factor Fli-1 interacts with SRF to form ternary complexes with this element. Two regions of Fli-1 that are required for ternary complex formation have been identified. These SRF binding motifs are located on either side of the ETS DNA-binding domain. Hydrophobic amino acids within these motifs have been identified that play important roles in binding to SRF and ternary complex formation. By using Fli-1 derivatives with mutations in the N-terminal SRF binding motif, the significance of Fli-1-SRF interactions in recruitment of Fli-1 to the c- fos SRE in vivo has been demonstrated. Collectively our data provide a model of how Fli-1 interacts with SRF that differs significantly from the mechanism used by a different ETS-domain protein, Elk-1.

Developmental regulation of V(D)J recombination at the TCR a/5 locus

The T-cell receptor (TCR) alpha/delta locus includes a large number of V, D, J and C gene segments that are used to produce functional TCR delta and TCR alpha chains expressed by distinct subsets of T lymphocytes. V(D)J recombination events within the locus are regulated as a function of developmental stage and cell lineage during T-lymphocyte differentiation in the thymus. The process of V(D)J recombination is regulated by cis-acting elements that modulate the accessibility of chromosomal substrates to the recombinase. Here we evaluate how the assembly of transcription factor complexes onto enhancers, promoters and other regulatory elements within the TCR alpha/delta locus imparts developmental control to VDJ delta and VJ alpha rearrangement events. Furthermore, we develop the notion that within a complex locus such as the TCR alpha/delta locus, highly localized and region-specific control is likely to require an interplay between positive regulatory elements and blocking or boundary elements that restrict the influence of the positive elements to defined regions of the locus.

CIITA stimulation of transcription factor binding to major histocompatibility complex class II and associated promoters in vivo.

CIITA is a master transactivator of the major histocompatibility complex class II genes, which are involved in antigen presentation. Defects in CIITA result in fatal immunodeficiencies. CIITA activation is also the control point for the induction of major histocompatibility complex class II and associated genes by interferon-gamma, but CIITA does not bind directly to DNA. Expression of CIITA in G3A cells, which lack endogenous CIITA, followed by in vivo genomic footprinting, now reveals that CIITA is required for the assembly of transcription factor complexes on the promoters of this gene family, including DRA, Ii, and DMB. CIITA-dependent promoter assembly occurs in interferon-gamma-inducible cell types, but not in B lymphocytes. Dissection of the CIITA protein indicates that transactivation and promoter loading are inseparable and reveal a requirement for a GTP binding motif. These findings suggest that CIITA may be a new class of transactivator.

TNF Regulates the In Vivo Occupancy of Both Distal and Proximal Regulatory Regions of the MCP-1/JE Gene

In vivo genomic footprinting (IVGF) was used to examine regulatory site occupancy during the activation of the murine inflammatory response gene MCP-1/JE by TNF. In response to TNF, both promoter distal and proximal regulatory regions became occupied in vivo. EMSA analysis showed that while some of the factors involved in expression, including NF-κB, were translocated to the nucleus following TNF treatment, others were already present and able to bind DNA in vitro. Protein kinase inhibitor studies showed that protein phosphorylation was required for TNF activation but not factor assembly. These studies provide evidence for a multistep model of TNF-mediated gene regulation involving chromatin accessibility, transcription factor complex assembly, and protein phosphorylation.

Lipopolysaccharide-mediated transcriptional activation of the human tissue factor gene in THP-1 monocytic cells requires both activator protein 1 and nuclear factor kappa B binding sites.

Lipopolysaccharide (LPS) activation of cells of monocytic lineage leads to rapid and transient expression of a set of inflammatory gene products, including tissue factor (TF). This transmembrane receptor is the major cellular initiator of the blood coagulation cascades, and induced expression of TF is postulated to play a role in inflammation. Functional studies using transfected THP-1 monocytic cells revealed the presence of a 56-bp LPS response element (LRE) within the TF promoter that conferred LPS responsiveness to a heterologous promoter. LPS stimulation of these cells activated proteins that bound to nucleotide sequences within the LRE resembling consensus binding sites for activator protein 1 (AP-1) and nuclear factor kappa B (NF-kappa B). Induction of the TF gene may represent a prototypic example of gene activation in monocytic cells by assembly of transcription factor complexes, and may clarify the role of AP-1 and NF-kappa B in the regulation of other LPS-responsive genes.