Isolation of novel coregulatory protein networks associated with DNA-bound estrogen receptor alpha

<p>Abstract</p> <p>Background</p> <p>DNA-bound transcription factors recruit an array of coregulatory proteins that influence gene expression. We previously demonstrated that DNA functions as an allosteric modulator of estrogen receptor α (ERα) conformation, alters the recruitment of regulatory proteins, and influences estrogen-responsive gene expression and reasoned that it would be useful to develop a method of isolating proteins associated with the DNA-bound ERα using full-length receptor and endogenously-expressed nuclear proteins.</p> <p>Results</p> <p>We have developed a novel approach to isolate large complexes of proteins associated with the DNA-bound ERα. Purified ERα and HeLa nuclear extracts were combined with oligos containing ERα binding sites and fractionated on agarose gels. The protein-DNA complexes were isolated and mass spectrometry analysis was used to identify proteins associated with the DNA-bound receptor. Rather than simply identifying individual proteins that interact with ERα, we identified interconnected networks of proteins with a variety of enzymatic and catalytic activities that interact not only with ERα, but also with each other. Characterization of a number of these proteins has demonstrated that, in addition to their previously identified functions, they also influence ERα activity and expression of estrogen-responsive genes.</p> <p>Conclusion</p> <p>The agarose gel fractionation method we have developed would be useful in identifying proteins that interact with DNA-bound transcription factors and should be easily adapted for use with a variety of cultured cell lines, DNA sequences, and transcription factors.</p

Interaction of estrogen receptor α with proliferating cell nuclear antigen

The ability of estrogen receptor α (ERα) to modulate gene expression is influenced by the recruitment of a host of co-regulatory proteins to target genes. To further understand how estrogen-responsive genes are regulated, we have isolated and identified proteins associated with ERα when it is bound to DNA containing the consensus estrogen response element (ERE). One of the proteins identified in this complex, proliferating cell nuclear antigen (PCNA), is required for DNA replication and repair. We show that PCNA interacts with ERα in the absence and in the presence of DNA, enhances the interaction of ERα with ERE-containing DNA, and associates with endogenous estrogen-responsive genes. Interestingly, rather than altering hormone responsiveness of endogenous, estrogen-responsive genes, PCNA increases the basal expression of these genes. Our studies suggest that in addition to serving as a platform for the recruitment of DNA replication and repair proteins, PCNA may serve as a platform for transcription factors involved in regulating gene expression

Histone H1 phosphorylation is associated with transcription by RNA polymerases I and II

Functional diversity of histone H1 variants may be caused by differences in phosphorylation during interphase

Two Regions Within the Proximal Steroidogenic Factor 1 Promoter Drive Somatic Cell-Specific Activity in Developing Gonads of the Female Mouse1

Targets of steroidogenic factor 1 (SF1; also known as NR5A1 and AD4BP) have been identified within cells at every level of the hypothalamic-pituitary-gonadal and -adrenal axes, revealing SF1 to be a master regulator of major endocrine systems. Mouse embryos express SF1 in the genital ridge until Embryonic Day 13.5 (E13.5). Thereafter, expression persists in the male and is substantially lower in the female gonad until birth. We hypothesize that the sexually dimorphic expression of Sf1 during gonadogenesis is mediated by sex-specific regulation of its promoter. To investigate dimorphic regulation within the fetal gonad, we developed an experimental strategy using transient transfection of E13.5 gonad explant cultures and evaluated various Sf1 promoter constructs for sexually dimorphic DNA elements. The proximal Sf1 promoter correctly targeted reporter activity to SF1-expressing cells in both XY and XX gonads. Stepwise deletion of sequences from the Sf1 promoter revealed two regions that affected regulation within female gonads. Mutation of both sequences together did not cause further disruption of reporter activity, suggesting the two sites might work in concert to promote activity in female somatic cells. Results from gel mobility shift assays and fetal gonad-chromatin immunoprecipitation showed that TCFAP2 binds to one of the two female-specific sites within the proximal promoter of Sf1. Together, we show that transient transfection experiments performed within developing testes and ovaries are a powerful tool to uncover elements within the Sf1 promoter that contribute to sex-specific expression

\u3ci\u3eDrosophila\u3c/i\u3e Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu