WD repeat-containing protein 5, a ubiquitously expressed histone methyltransferase adaptor protein, regulates smooth muscle cell-selective gene activation through interaction with pituitary homeobox 2

WD repeat-containing protein 5 (WDR5) is a common component of mammalian mixed lineage leukemia methyltransferase family members and is important for histone H3 lysine 4 methylation (H3K4me), which has been implicated in control of activation of cell lineage genes during embryogenesis. However, WDR5 has not been considered to play a specific regulatory role in epigenetic programming of cell lineage because it is ubiquitously expressed. Previous work from our laboratory showed the appearance of histone H3K4me within smooth muscle cell (SMC)-marker gene promoters during the early stages of development of SMC from multipotential embryonic cells but did not elucidate the underlying mechanisms that mediate SMC-specific and locus-selective H3K4me. Results presented herein show that knockdown of WDR5 significantly decreased SMC-marker gene expression in cultured SMC differentiation systems and in Xenopus laevis embryos in vivo. In addition, we showed that WDR5 complexes within SMC progenitor cells contained H3K4 methyltransferase enzymatic activity and that knockdown of WDR5 selectively decreased H3K4me1 and H3K4me3 enrichment within SMC-marker gene promoter loci. Moreover, we present evidence that it is recruited to these gene promoter loci through interaction with a SMC-selective pituitary homeobox 2 (Pitx2). Taken together, studies provide evidence for a novel mechanism for epigenetic control of SMC-marker gene expression during development through interaction of WDR5, homeodomain proteins, and chromatin remodeling enzymes.     

Differential requirement for steroidogenic factor-1 gene dosage in adrenal development versus endocrine function

The importance of steroidogenic factor-1 (SF-1) gene dosage in endocrine function is evidenced by phenotypes associated with the heterozygous state in mice and humans. Here we examined mechanisms underlying SF-1 haploinsufficiency and found a striking reduction (12-fold) in SF-1 heterozygous (+/-) adrenocortical size at embryonic day (E) 12. Loss of one SF-1 allele led to a selective decrease in adrenal precursors within the adrenogonadal primordium at E10.0, without affecting the number of gonadal precursors, as marked by GATA-4. Beginning at E13.5, increased cell proliferation in SF-1 +/- adrenals allows these organs to approach but not attain a normal size. Remarkably, neural crest-derived adrenomedullary precursors migrated normally in SF-1 +/- and null embryos. However, later in development, medullary growth was compromised in both genotypes. Despite the small adrenal size in SF-1heterozygotes, an unexpected elevation in steroidogenic capacity per cell was observed in primary adult adrenocortical SF-1 +/- cells compared with wild-type cells. Elevated cellular steroid output is consistent with the up-regulation of some SF-1 target genes in SF-1 +/- adrenals and may partially be due to an observed increase in nerve growth factor-induced-B. Our findings underscore the need for full SF-1 gene dosage early in adrenal development, but not in the adult adrenal, where compensatory mechanisms restore near normal function.     

Proteomic analysis of nuclear factors binding to an intronic enhancer in the myelin proteolipid protein gene

The myelin proteolipid protein gene ( Plp1 ) encodes the most abundant protein found in CNS myelin, accounting for nearly one-half of the total protein. Its expression in oligodendrocytes is developmentally regulated – peaking during the active myelination period of CNS development. Previously, we have identified a novel enhancer (designated ASE) in intron 1 DNA that appears to be important in mediating the surge of Plp1 gene activity during the active myelination period. Evidence suggests that the ASE participates in the formation of a specialized multi-protein/DNA complex called an enhanceosome. The current study describes an optimized, five-step, DNA affinity chromatography purification procedure to purify nuclear proteins from mouse brain that bind to the 85-bp ASE sequence, specifically. Electrophoretic mobility shift assay analysis demonstrated that specific DNA-binding activity was retained throughout the purification procedure, resulting in concomitant enrichment of nucleoprotein complexes. Identification of the purported regulatory factors was achieved through mass spectrometry analysis and included over 20 sequence-specific DNA-binding proteins. Supplementary western blot analyses to determine which of these sequence-specific factors are present in oligodendrocytes, and their developmental and regional expression in whole brain, suggest that Purα and Purβ rank highest among the candidate factors as constituents of the multi-protein complex formed on the ASE.     

A TEAD1/p65 complex regulates the eutherian-conserved MnSOD intronic enhancer,eRNA transcription and the innate immune response

Manganese superoxide dismutase (MnSOD), a critical anti-oxidant enzyme, detoxifies the mitochondrial-derived reactive oxygen species, superoxide, elicited through normal respiration or the inflammatory response. Proinflammatory stimuli induce MnSOD gene expression through a eutherian-conserved, intronic enhancer element. We identified two prototypic enhancer binding proteins, TEAD1 and p65, that when co-expressed induce MnSOD expression comparable to pro-inflammatory stimuli. TEAD1 causes the nuclear sequestration of p65 leading to a novel TEAD1/p65 complex that associates with the intronic enhancer and is necessary for cytokine induction of MnSOD. Unlike typical NF-κB-responsive genes, the induction of MnSOD does not involve p50. Beyond MnSOD, the TEAD1/p65 complex regulates a subset of genes controlling the innate immune response that were previously viewed as solely NF-κB-dependent. We also identified an enhancer-derived RNA (eRNA) that is induced by either proinflammatory stimuli or the TEAD1/p65 complex, potentially linking the intronic enhancer to intra- and interchromosomal gene regulation through the inducible eRNA.