Subir K. Ray

Basic helix loop helix transcription factors and enteroendocrine cell differentiation

For over 30 years it has been known that enteroendocrine cells derive from common precursor cells in the intestinal crypts. Until recently little was understood about the events that result in commitment to endocrine differentiation or the eventual segregation of over 10 different hormone‐expressing cell types in the gastrointestinal tract. Enteroendocrine cells arise from pluripotent intestinal stem cells. Differentiation of enteroendocrine cells is controlled by the sequential expression of three basic helix‐loop‐helix transcription factors, Math1, Neurogenin 3 (Neurog3) and NeuroD. Math1 expression is required for specification and segregation of the intestinal secretory lineage (Paneth, goblet,and enteroendocrine cells) from the absorptive enterocyte lineage. Neurog3 expression represents the earliest stage of enteroendocrine differentiation and in its absence enteroendocrine cells fail to develop. Subsequent expression of NeuroD appears to represent a later stage of differentiation for maturing enteroendocrine cells. Enteroendocrine cell fate is inhibited by the Notch signalling pathway, which appears to inhibit both Math1 and Neurog3. Understanding enteroendocrine cell differentiation will become increasingly important for identifying potential future targets for common diseases such as diabetes and obesity.

Energy Homeostasis and Gastrointestinal Endocrine Differentiation Do Not Require the Anorectic Hormone Peptide YY

ABSTRACT The gastrointestinal hormone peptide YY is a potent inhibitor of food intake and is expressed early during differentiation of intestinal and pancreatic endocrine cells. In order to better understand the role of peptide YY in energy homeostasis and development, we created mice with a targeted deletion of the peptide YY gene. All intestinal and pancreatic endocrine cells developed normally in the absence of peptide YY with the exception of pancreatic polypeptide (PP) cells, indicating that peptide YY expression was not required for terminal differentiation. We used recombination-based cell lineage trace to determine if peptide YY cells were progenitors for gastrointestinal endocrine cells. Peptide YY+ cells gave rise to all L-type enteroendocrine cells and to islet ∂ and PP cells. In the pancreas, approximately 40% of pancreatic α and rare β cells arose from peptide YY+ cells, suggesting that most β cells and surprisingly the majority of α cells are not descendants of peptide YY+/glucagon-positive/insulin-positive cells that appear during early pancreagenesis. Despite the anorectic effects of exogenous peptide YY3-36 following intraperitoneal administration, mice lacking peptide YY showed normal growth, food intake, energy expenditure, and responsiveness to peptide YY3-36. These observations suggest that targeted disruption of the peptide YY gene does not perturb terminal endocrine cell differentiation or the control of food intake and energy homeostasis.

Novel Transcriptional Potentiation of BETA2/NeuroD on the Secretin Gene Promoter by the DNA-Binding Protein Finb/RREB-1

ABSTRACT The basic helix-loop-helix protein BETA2/NeuroD activates transcription of the secretin gene and is essential for terminal differentiation of secretin-producing enteroendocrine cells. However, in heterodimeric complexes with its partner basic helix-loop-helix proteins, BETA2 does not appear to be a strong activator of transcription by itself. Mutational analysis of a proximal enhancer in the secretin gene identified several cis-acting elements in addition to the E-box binding site for BETA2. We identified by expression cloning the zinc finger protein RREB-1, also known to exist as a longer form, Finb, as the protein binding to one of the mutationally sensitive elements. Finb/RREB-1 lacks an intrinsic activation domain and by itself did not activate secretin gene transcription. Here we show that Finb/RREB-1 can associate with BETA2 to enhance its transcription-activating function. Both DNA binding and physical interaction of Finb/RREB-1 with BETA2 are required to potentiate transcription. Thus, Finb/RREB-1 does not function as a classical activator of transcription that recruits an activation domain to a DNA-protein complex. Finb/RREB-1 may be distinguished from coactivators, which increase transcription without sequence-specific DNA binding. We suggest that Finb/RREB-1 should be considered a potentiator of transcription, representing a distinct category of transcription-regulating proteins.

CtBP and Associated LSD1 Are Required for Transcriptional Activation by NeuroD1 in Gastrointestinal Endocrine Cells

ABSTRACT Gene expression programs required for differentiation depend on both DNA-bound transcription factors and surrounding histone modifications. Expression of the basic helix-loop-helix (bHLH) protein NeuroD1 is restricted to endocrine cells in the gastrointestinal (GI) tract, where it is important for endocrine differentiation. RREB1 (RAS-responsive element binding protein 1), identified as a component of the CtBP corepressor complex, binds to nearby DNA elements to associate with NeuroD and potentiate transcription of a NeuroD1 target gene. Transcriptional activation by RREB1 depends on recruitment of CtBP with its associated proteins, including LSD1, through its PXDLS motifs. The mechanism of transcriptional activation by CtBP has not been previously characterized. Here we found that activation was dependent on the histone H3 lysine 9 (H3K9) demethylase activity of LSD1, which removes repressive methyl marks from dimethylated H3K9 (H3K9Me2), to facilitate subsequent H3K9 acetylation by the NeuroD1-associated histone acetyltransferase, P300/CBP-associated factor (PCAF). The secretin, β-glucokinase, insulin I, and insulin II genes, four known direct targets of NeuroD1 in intestinal and pancreatic endocrine cells, all show similar promoter occupancy by CtBP-associated proteins and PCAF, with acetylation of H3K9. This work may indicate a mechanism for selective regulation of transcription by CtBP and LSD1 involving their association with specific transcription factors and cofactors to drive tissue-specific transcription.

The Basic Helix-Loop-Helix Transcription Factor NeuroD1 Facilitates Interaction of Sp1 with the Secretin Gene Enhancer

ABSTRACT The basic helix-loop-helix transcription factor NeuroD1 is required for late events in neuronal differentiation, for maturation of pancreatic β cells, and for terminal differentiation of enteroendocrine cells expressing the hormone secretin. NeuroD1-null mice demonstrated that this protein is essential for expression of the secretin gene in the murine intestine, and yet it is a relatively weak transcriptional activator by itself. The present study shows that Sp1 and NeuroD1 synergistically activate transcription of the secretin gene. NeuroD1, but not its widely expressed dimerization partner E12, physically interacts with the C-terminal 167 amino acids of Sp1, which include its DNA binding zinc fingers. NeuroD1 stabilizes Sp1 DNA binding to an adjacent Sp1 binding site on the promoter to generate a higher-order DNA-protein complex containing both proteins and facilitates Sp1 occupancy of the secretin promoter in vivo. NeuroD-dependent transcription of the genes encoding the hormones insulin and proopiomelanocortin is potentiated by lineage-specific homeodomain proteins. The stabilization of binding of the widely expressed transcription factor Sp1 to the secretin promoter by NeuroD represents a distinct mechanism from other NeuroD target genes for increasing NeuroD-dependent transcription.

Review article: transcriptional events controlling the terminal differentiation of intestinal endocrine cells

Secretin‐producing enteroendocrine cells arise from a multipotential endocrine progenitor in the crypts of the small intestine. As these cells migrate up the crypt‐villus axis, they produce secretin and stop dividing as they terminally differentiate and die. Transcription of the secretin gene is controlled by a complex enhancer binding to multiple transcription factors. The basic helix‐loop‐helix protein, BETA2, binds to an E box sequence and associates with the p300 coactivator to activate transcription of the secretin gene. Basic helix‐loop‐helix proteins appear to play a pivotal role in the control of cellular differentiation. BETA2 induces cell cycle arrest and apoptosis in addition to activating secretin gene expression. Thus BETA2 may function as a master regulatory gene to coordinate terminal differentiation of secretin cells.

Oligomeric form of C-terminal-binding protein coactivates NeuroD1-mediated transcription

The mechanism underlying transcriptional coactivation by the corepressor C‐terminal‐binding protein (CtBP) is not established. We previously found that CtBP co‐occupies several actively transcribed endocrine genes with the transcription factor NeuroD1 to paradoxically increase transcription by recruiting KDM1A and CoREST. While the importance of the oligomeric form of CtBP for corepression is well established, the role of oligomerization in transcriptional coactivation has received little attention. Here, we examined the importance of the oligomeric state of CtBP for coactivation of NeuroD1‐dependent transcription by expressing a CtBP dimerization mutant in cells depleted of endogenous CtBP. Dimerization mutants failed to increase transcription or to associate with KDM1A and CoREST, suggesting that oligomeric, but not monomeric CtBP is required to recruit other proteins needed to activate transcription.

Sp1 and finb interact with BETA2/NeuroD to enhance its transactivation of the secretin promoter

of full length fragment which covers all three gastrin responsive elements were cloned for yeast one hybrid screening. Electrophoretic mobility shift assays (EMSAs) were then used to confirm the positives. To determine the functional importance of the target factor(s), transient transfection and reporter assays were performed by overexpression of the target nuclear factor(s) in the presence of the HDC promoter construct which contains 125bp upstream sequence and 126bp downstream sequence followed by the luciferase gene. Results: Yeast one hybrid screening indicated that gut-enriched Kruppel like factor (GKLF) binds downstream gastrin responsive elements of the HDC promoter. Electrophoretic mobility shift assays confirmed the binding of GKLF with all three downstream gastrin responsive elements. EMSA competition assay also showed the binding of Spl with all three gastrin responsive elements. Furthermore, EMSAs indicated that GKLF binds Spl bound GC-box fragment which is located upstream of the HDC promoter. Overexpression of GKLF and Spl transactivated HDC promoter activity, which was further supported by the repressive effect o f their dominant negative plasmids. Conclusion: GKLF and Sp 1 synergistically regulate HDC promoter activity through binding both upstream GC box and downstream gastrin responsive elements.