Identification and Validation of a Putative Polycomb Responsive Element in the Human Genome.

Epigenetic cellular memory mechanisms that involve polycomb and trithorax group of proteins are well conserved across metazoans. The cis-acting elements interacting with these proteins, however, are poorly understood in mammals. In a directed search we identified a potential polycomb responsive element with 25 repeats of YY1 binding motifthatwe designate PRE-PIK3C2B as it occurs in the first intron of human PIK3C2B gene. It down regulates reporter gene expression in HEK cells and the repression is dependent on polycomb group of proteins (PcG). We demonstrate that PRE-PIK3C2B interacts directly with YY1 in vitro and recruits PRC2 complex in vivo. The localization of PcG proteins including YY1 to PRE-PIK3C2B in HEK cells is decreased on knock-down of either YY1 or SUZ12. Endogenous PRE-PIK3C2B shows bivalent marking having H3K27me3 and H3K4me3 for repressed and active state respectively. In transgenic Drosophila, PRE-PIK3C2B down regulates mini-white expression, exhibits variegation and pairing sensitive silencing (PSS), which has not been previously demonstrated for mammalian PRE. Taken together, our results strongly suggest that PRE-PIK3C2B functions as a site of interaction for polycomb proteins.

YY1 negatively regulates mouse myelin proteolipid protein (Plp1) gene expression in oligodendroglial cells.

YY1 (Yin and Yang 1) is a multifunctional, ubiquitously expressed, zinc finger protein that can act as a transcriptional activator, repressor, or initiator element binding protein. Previous studies have shown that YY1 modulates the activity of reporter genes driven by the myelin PLP (proteolipid protein) (PLP1/Plp1) promoter. However, it is known that Plp1 intron 1 DNA contains regulatory elements that are required for the dramatic increase in gene activity, coincident with the active myelination period of CNS (central nervous system) development. The intron in mouse contains multiple prospective YY1 target sites including one within a positive regulatory module called the ASE (anti-silencer/enhancer) element. Results presented here demonstrate that YY1 has a negative effect on the activity of a Plp1-lacZ fusion gene [PLP(+)Z] in an immature oligodendroglial cell line (Oli-neu) that is mediated through sequences present in Plp1 intron 1 DNA. Yet YY1 does not bind to its alleged site in the ASE (even though the protein is capable of recognizing a target site in the promoter), indicating that the down-regulation of PLP(+)Z activity by YY1 in Oli-neu cells does not occur through a direct interaction of YY1 with the ASE sequence. Previous studies with Yy1 conditional knockout mice have demonstrated that YY1 is essential for the differentiation of oligodendrocyte progenitors. Nevertheless, the current study suggests that YY1 functions as a repressor (not an activator) of Plp1 gene expression in immature oligodendrocytes. Perhaps YY1 functions to keep the levels of PLP in check in immature cells before vast quantities of the protein are needed in mature myelinating oligodendrocytes.

[Structure characteristics of the chloroplast rpS16 intron in Allium sativum and related Allium species].

For the first time the chloroplast rpS16 intron sequences in A. sativum accessions with different ecologo-geographical origins and related Allium species have been characterized. The main stem-loop consensus sequences and boundaries ofsix domains have been identified and the most probable secondary structure model of the intron pre-RNA has been predicted. Allium rpS16 introns have been characterized by mutation rate heterogeneity between structure regions of all six domains. Domains II and IV of the intron are shown to be more variable with transition predominance in I, III, V and VI domain sequences. In addition to structure elements typical for group IIB introns the Allium specific micro- and macrostructural alterations have been revealed. The 290 nucleotide deletion of domains III-IV sequences and of the part of the domain V has been revealed in A. altaicum, A. fistulosum, A. schoenoprasum rpS16 intron sequences. Several nucleotide substitutions and extensive length mutations result in secondary structure deviation from the consensus model of group II introns.

Cooperative actions of Tra2alpha with 9G8 and SRp30c in the RNA splicing of the gonadotropin-releasing hormone gene transcript.

In earlier studies, we demonstrated that excision of the first intron (intron A) from the gonadotropin-releasing hormone (GnRH) transcript is highly cell type- and developmental stage-specific. The removal of GnRH intron A requires exonic splicing enhancers on exons 3 and 4 (ESE3 and ESE4, respectively). Tra2alpha,a serine/arginine-rich (SR)-like protein, specifically binds to ESE4, although it requires additional nuclear co-factors for efficient removal of this intron. In the present study, we demonstrate the cooperative action of multiple SR proteins in the regulation of GnRH pre-mRNA splicing. SRp30c specifically binds to both ESE3 and ESE4, whereas 9G8 binds to an element in exon 3 and strongly enhances the excision of GnRH intron A in the presence of minimal amount of other nuclear components. Interestingly, Tra2alpha can interact with either 9G8 or SRp30c, whereas no interaction between 9G8 and SRp30c is observed. Tra2alpha has an additive effect on the RNA binding of these proteins. Overexpression or knock-down of these three proteins in cultured cells further suggests their essential role in intron A excision activities, and their presence in GnRH neurons of the mouse preoptic area further strengthens this possibility. Together, these results indicate that interaction of Tra2alpha with 9G8 and SRp30c appears to be crucial for ESE-dependent GnRH pre-mRNA splicing, allowing efficient generation of mature mRNA in GnRH-producing cells.

GATA2 is required for the generation of V2 interneurons.

During embryogenesis, transcription factor GATA2 is expressed in a variety of distinct cell types, and earlier experiments showed that GATA2 is a vital regulator of both hematopoiesis and urogenital development. Despite the fact that GATA2 is expressed early and abundantly in the nervous system, there has been no demonstration of its direct participation in neurogenesis. We show here that GATA2 is expressed in the ventral spinal cord exclusively in newly generated V2 interneurons, suggesting that GATA2 might be required for the generation of this discrete neuronal population. Proof for this hypothesis was provided by showing that the number of cells expressing V2 neuronal markers was drastically diminished in gata2 null mutant embryos. The tissue-specific enhancer that directs gata2 transcription specifically in V2 neurons was localized to a 190 bp intragenic element lying within gata2 intron 5, and this element is both necessary and sufficient to confer GATA2 spinal cord expression. The identification of a V2-specific enhancer should allow fundamental new insight into the genetic hierarchy of regulatory events that govern neurogenesis in a well-defined cell lineage.

Bidirectional transcription regulation of glial fibrillary acidic protein by estradiol in vivo and in vitro.

Glial fibrillary acidic protein (GFAP) expression shows cyclic variation in the rat hypothalamus and hippocampus during the normal estrous cycle. To elucidate the role of transcription in the regulation of GFAP, we examined levels of GFAP intron 1 by in situ hybridization in the hypothalamus and hippocampus of normal, cycling rats. On the afternoon of proestrus, when plasma estradiol levels are highest, GFAP transcription and messenger RNA were both increased in the arcuate nucleus of the hypothalamus and decreased in the outer molecular layer of the dentate gyrus. In the hilus of the hippocampus, neither GFAP transcription nor messenger RNA changed during the estrous cycle. In vitro, astrocytes showed bidirectional responses, such that estradiol treatment increased GFAP transcription in monotypic astrocytic cultures but decreased GFAP transcription in astrocytes cocultured with neurons. The functionality of an estrogen response element in the 5'-upstream region of the GFAP promoter was established by site-directed mutagenesis and binding of human recombinant estrogen receptor in gel shift assays. We conclude that estrogen may act directly upon astrocytes by estrogen receptor binding, and that the direction of the transcriptional response is influenced by astrocyte-neuron interactions.

Unusual proopiomelanocortin ribonucleic acids in extrapituitary tissues: intronless transcripts in testes and long poly(A) tails in hypothalamus.

The POMC gene is predominantly expressed in the pituitary gland; it is also expressed in various extrapituitary tissues. While POMC mRNAs of similar size (approximately equal to 1000 nucleotides) are present in the anterior and neurointermediate lobes of the pituitary, other POMC-expressing tissues contain POMC mRNAs of different sizes. Longer POMC mRNAs are observed in the hypothalamus. Using S1 nuclease mapping and mRNA deadenylation by RNase H, we have shown that these large hypothalamic POMC mRNAs have longer poly(A) tails than pituitary POMC transcripts but contain the same transcripted sequences. In contrast, the testes contain POMC transcripts which are smaller than pituitary POMC mRNA. RNase and S1 nuclease mapping analyses suggest that these short transcripts do not contain sequences transcribed from pituitary exons 1 and 2. Indeed, as revealed by primer-extension experiments, these transcripts appear to initiate within exon 3 sequences of the POMC gene. The heterogeneous 5'-ends of these short testicular transcripts map into the NH2-terminal portion of the precursor in the region encoding gamma MSH; if ever translated, these transcripts would produce a form of POMC that would be truncated at the NH2-terminus and therefore would be devoid of any signal peptide sequence. Interestingly, the sequence of the short testicular transcripts corresponds to that of the mouse POMC pseudogene, suggesting that this POMC pseudogene may have derived from genomic integration of testicular transcripts via a cDNA intermediate.