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Fox A1


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Fox A1

  1. 2. <ul><li>Forkhead box (Fox) proteins are a family of evolutionarily conserved transcriptional regulators defined by a common DNA-binding domain (DBD) termed the forkhead box or winged helix domain. </li></ul><ul><li>The first protein in the FOX family that was discovered was the fork head transcription factor in Drosophila (thus the name). </li></ul><ul><li>The forkhead factors bind to chromatin structure highly compacted (silenced), remodeling and generating a &quot;permissive&quot; environment for the binding of other transcription factors. </li></ul><ul><li>Fox protein regulation and function vary significantly between families, arising in part from sequence variation outside of the DBD, allowing for differential regulation and functional diversification </li></ul>Model for FOXA mechanism. This factor contains a Fkh domain that may antagonize H1-mediated repression by their ability to “open-up” chromatin arrays repressed with histone H1. The winged helix domain of a Fkh factor is shown associated with DNA helix on the edge of a nucleosome. Forkhead box (Fox) proteins
  2. 3. INTRODUCTION <ul><li>Pluripotent cell  differentiated cell lineage </li></ul><ul><li>Cell type-specific transcriptional programs </li></ul><ul><li>FoxA1 (Hepatocyte Nuclear Factor 3a – HNF3a) – development and differentiation of liver, kidney, pancreas, lung, prostate and mammary gland </li></ul><ul><li>High expresion in prostate and ERa+ breast tumors (positive prognostic factor, sensitivity to endocrine therapy) </li></ul><ul><li>FoxA1 – a pioneer factor in the recruitment of ERa to several cis-regulatory elements  transcriptional induction of E2 regulated genes in breast cancer cells </li></ul><ul><li>FoxA1 – androgen receptor (AR) in prostate cancer cells – impacts regulation of AR target genes </li></ul><ul><li>FoxA1 regulates distinct transcriptional programs in cells of different lineage </li></ul><ul><li>¿molecular basis for differential transcriptional activities? </li></ul><ul><li>GOAL: to investigate FoxA1 differential transcriptional activities in breast and prostate cancer cells in relation to their epigenome </li></ul> Genome –wide positional analysis ChIP-chip
  3. 4. RESULTS <ul><li>Dual Regulatory Role of FoxA1 in E2 Signaling </li></ul>E2 stimulation  ERa (+) breast cancer cells  specific transcriptional program <ul><li>¿How does FoxA1 participate in this proces? </li></ul><ul><li>unbiased genome-wide ChIP-chip study using tilling microarrays </li></ul><ul><ul><li>Defined FoxA1 binding sites - “cistrome” in MCF7 breast cancer cell line </li></ul></ul><ul><ul><li>Defined ER a cistrome </li></ul></ul><ul><li>Conclusions: </li></ul><ul><ul><li>FoxA1 binding sites are distant from the proximal promotor regions of genes </li></ul></ul><ul><ul><li>50-60% overlap of FoxA1 and ER a binding sites  co-binding </li></ul></ul>
  4. 5. <ul><li>¿Functional significance of this co-binding? </li></ul><ul><li> comparing the fraction of E2 regulated genes and E2 non-regulated genes in respect to ERa unique, FoxA1 unique, both ERa and FoxA1, and overlaping ERa /FoxA1 binding sites near their TSS </li></ul><ul><li>More E2 up-regulated genes with ERa /FoxA1 overlaping binding site than non-E2 regulated genes </li></ul><ul><li>More E2 down-regulated genes with FoxA1 only and ERa /FoxA1 overlaping binding site than non-E2 regulated genes </li></ul>Genes with FoxA1-binding sites within 20 kb of their TSS also had a greater chance to be expressed together with FoxA1 and ERa in primary breast tumors pointing to the biological relevance of the FoxA1 cistrome beyond the MCF7 cell line
  5. 6. FoxA1 Cell Type-Specific Activity Depends on Differential Recruitment to Chromatin <ul><li>¿How does FoxA1 binding to chromatin relates to its cell specific functions? </li></ul><ul><li> Compared FoxA1 cistromes from MCF7 breast cancer and LNCaP prostate cancer cell lines </li></ul><ul><li>The majority (65%) of Fox A1 recruitment sites is cell type specific </li></ul><ul><li>FoxA1 might regulate differential transcriotional programs as a result of cell type specific recruitment pattern </li></ul><ul><li>¿association of FoxA1-binding sites unique to MCF7 or LNCaP, or sites shared between the two cell lines, with genes coexpressed with FoxA1 in primary breast of prostate tumors </li></ul>Differential recruitment is the primary mechanism responsible for the differential function of FoxA1 in these two different cell lineages.
  6. 7. FoxA1 Alternatively Collaborates with ERa or AR at Cell-Specific Enhancers <ul><li>¿ Correlation between E2 or DHT regulated genes and binding sites for FoxA1 and ERa in MCF7 cells or for FoxA1 and AR in LNCaP cells? </li></ul> Transcription factor binding motifs enriched within the common FoxA1 recruitment sites, as well as those unique to each cell line <ul><li>the Forkhead motif (FKHR) was enriched in all three subsets of FoxA1-binding regions </li></ul><ul><li>recognition motifs for the nuclear receptors ERa (ERE and ERE half-site) and AR (ARE and ARE half-site) were specifically enriched in FoxA1-binding sites unique to MCF7 or to </li></ul><ul><li>LNCaP cells </li></ul><ul><li>genes regulated by E2 were significantly enriched over nonregulated genes near ERa </li></ul><ul><li>sites overlapping with FoxA1 in MCF7 cells </li></ul><ul><li>genes regulated by DHT were specifically significantly enriched over nonregulated genes near AR sites overlapping with FoxA1 in LNCaP cells </li></ul>
  7. 8. A Cell Type-Specific Histone Signature Correlates with Differential FoxA1 Recruitment <ul><li>¿how is FoxA1 able to bind to distinct regions within the genome of the MCF7 and LNCaP cells? </li></ul><ul><li>sequence recognized by FoxA1 could be different between the two cell lines? </li></ul><ul><ul><li>de novo motif analysis </li></ul></ul><ul><ul><li>No signicicant differences between recognition sequences </li></ul></ul><ul><li>differential FoxA1 binding could rather be linked to specific epigenetic modifications? </li></ul><ul><ul><ul><ul><li> Analyzing the presence of these specific histone </li></ul></ul></ul></ul><ul><ul><ul><ul><li>modifications at the FoxA1 recruitment sites revealed significant enrichment for H3K4me1 and me2 in a cell type-specific manner </li></ul></ul></ul></ul><ul><li> in MCF7 cells, FoxA1-binding sites unique to MCF7 cells as well as sites common to both cell lines were significantly mono- and dimethylated on H3K4 compared to the LNCaP unique FoxA1-binding sites </li></ul><ul><li> in LNCaP cells, the LNCaP-specific FoxA1- binding sites together with the common sites were significantly enriched for these histone modifications compared to MCF7-specific sites </li></ul>These results suggest a link between FoxA1 recruitment with the presence of H3K4me1 and me2.
  8. 9. FoxA1 Is Required for Chromatin Remodeling but Not for H3K4 Methylation FoxA1 silencing decreases chromatin accessibility of enhancers but not H3K4 methylation levels ¿Is H3K4me1 and me2 required for FoxA1 recruitment or is it induced as a result of FoxA1 binding to the chromatin?  DNAse protectivity assay - does FoxA1silencing affect H3K4 methylation, chromatin remodeling, or both in MCF7and LNCaPcells? <ul><li>FoxA1 silencing impacted the DNase I sensitivity only at those sites to which it was recruited </li></ul><ul><li>T hese sites did not in general show a significant reduction in the levels of H3K4me1 or me2 in enhancers in either MCF7 or LNCaP cells, but it affected the transcriptional regulation of </li></ul><ul><li>their target genes </li></ul>
  9. 10. Reduction of H3K4 Methylation Impairs Cell Type-Specific FoxA1 Recruitment ¿Effect of H3K4 mono- or dimethylation on cell type-specific recruitment of FoxA1?  overexpressed the H3K4me1 and me2 specific demethylase KDM1 in MCF7 cells and established its impact on FoxA1 recruitment <ul><li>H3K4me1 was slightly reduced and H3K4me2 was significantly lowered on FoxA1-binding sites </li></ul><ul><li>level of H3K9me2 remained unchanged at these sites </li></ul><ul><li>FoxA1 protein levels were unaffected by KDM1 overexpression but its recruitment to the chromatin was significantly impaired </li></ul><ul><li>H3K4me2 is required to define the cell type specific </li></ul><ul><li>regions competent for recruitment of FoxA </li></ul>
  10. 11. CONCLUSIONS <ul><li>FoxA1 diferential recruitment to chromatin at distant enhancers </li></ul><ul><li>Cell type specific changes in chromatin structure </li></ul><ul><li>Histone H3K4 methylation </li></ul><ul><li>Collaboration with lineage specific TF </li></ul>FoxA1 differential transcriptional activities in breast and prostate cells rely primarily on its differential recruitment to the chromatin and alternative collaboration with the lineage-specific factors ERa or AR at cell-specific enhancers Alternative transcriptional programs depend both on the orchestrated expression of a particular set of collaborating transcription factors together with their ability to bind cell specific enhancer elements in the vicinity of their target genes.