A novel DNMT3B splice variant expressed in tumor and pluripotent cells modulates genomic DNA methylation patterns and displays altered DNA binding.

DNA methylation is an epigenetic mark essential for mammalian development, genomic stability, and imprinting. DNA methylation patterns are established and maintained by three DNA methyltransferases: DNMT1, DNMT3A, and DNMT3B. Interestingly, all three DNMTs make use of alternative splicing. DNMT3B has nearly 40 known splice variants expressed in a tissue- and disease-specific manner, but very little is known about the role of these splice variants in modulating DNMT3B function. We describe here the identification and characterization of a novel alternatively spliced form of DNMT3B lacking exon 5 within the NH(2)-terminal regulatory domain. This variant, which we term DNMT3B3Delta5 because it is closely related in structure to the ubiquitously expressed DNMT3B3 isoform, is highly expressed in pluripotent cells and brain tissue, is downregulated during differentiation, and is conserved in the mouse. Creation of pluripotent iPS cells from fibroblasts results in marked induction of DNMT3B3Delta5. DNMT3B3Delta5 expression is also altered in human disease, with tumor cell lines displaying elevated or reduced expression depending on their tissue of origin. We then compared the DNA binding and subcellular localization of DNMT3B3Delta5 versus DNMT3B3, revealing that DNMT3B3Delta5 possessed significantly enhanced DNA binding affinity and displayed an altered nuclear distribution. Finally, ectopic overexpression of DNMT3B3Delta5 resulted in repetitive element hypomethylation and enhanced cell growth in a colony formation assay. Taken together, these results show that DNMT3B3Delta5 may play an important role in stem cell maintenance or differentiation and suggest that sequences encoded by exon 5 influence the functional properties of DNMT3B

Expression profiling after activation of amino acid deprivation response in HepG2 human hepatoma cells

Dietary protein malnutrition is manifested as amino acid deprivation of individual cells, which activates an amino acid response (AAR) that alters cellular functions, in part, by regulating transcriptional and posttranscriptional mechanisms. The AAR was activated in HepG2 human hepatoma cells, and the changes in mRNA content were analyzed by microarray expression profiling. The results documented that 1,507 genes were differentially regulated by P < 0.001 and by more than twofold in response to the AAR, 250 downregulated and 1,257 upregulated. The spectrum of altered genes reveals that amino acid deprivation has far-reaching implications for gene expression and cellular function. Among those cellular functions with the largest numbers of altered genes were cell growth and proliferation, cell cycle, gene expression, cell death, and development. Potential biological relationships between the differentially expressed genes were analyzed by computer software that generates gene networks. Proteins that were central to the most significant of these networks included c-myc, polycomb group proteins, transforming growth factor β1, nuclear factor (erythroid-derived 2)-like 2-related factor 2, FOS/JUN family members, and many members of the basic leucine zipper superfamily of transcription factors. Although most of these networks contained some genes that were known to be amino acid responsive, many new relationships were identified that underscored the broad impact that amino acid stress has on cellular function

ChIP analysis of the <i>Mkrn3</i> and <i>Ndn</i> promoters.

<p>A) ChIP analysis of the <i>Mkrn3</i> locus. Antibodies against NRF-2, Sp1, and YY1 were used to immunoprecipitate chromatin from the maternal and paternal alleles separately in Tg^PWSdel and Tg^ASdel mouse fibroblasts, respectively. The location of primers used to examine transcription factor binding within regions 1–4 across the <i>Mkrn3</i> locus are described further in the main text. The solid rectangle depicts the intronless <i>Mkrn3</i> gene; the bent arrow represents the transcription initiation site. Open bars represent analysis of the maternal allele, bars with horizontal stripes represent control samples from Tg^PWSdel cells (maternal allele) treated with no antibody, solid bars represent analysis of the paternal allele, and bars with vertical stripes represent control samples from Tg^ASdel cells (paternal allele) treated with no antibody. B) ChIP analysis of the <i>Mkrn3</i> promoter region (region 2 in panel A) in primary mouse brain and spleen cells. Brain and spleen cell preparations from C57BL/6 mice were subjected to ChIP analysis with antibodies against YY1, NRF-2, or RNA polymerase II. C) ChIP analysis of the <i>Ndn</i> promoter region in Tg^PWSdel and Tg^ASdel cells using antibodies against NRF-1, YY1, and Sp1.</p

ChIP analysis of transcription factor binding to the <i>Snrpn</i> locus.

<p>Chromatin immunoprecipitation analysis was performed to assay for binding of NRF-1 and YY1 to DH sites in the <i>Snrpn</i> 5′ region in primary brain and spleen cells of C57Bl/6 mice. A) Diagram of the mouse <i>Snrpn</i> 5′ region showing the regions assayed by ChIP. Pairs of opposing horizontal arrows depict the location of PCR primer sets used for real-time PCR in ChIP assays. Primer sets 1 and 3 are negative controls and amplify regions where no known factors are thought to be bound. B) Results of ChIP assays performed with antibodies against the transcription factors NRF-1 and YY1. Black and gray vertical bars denote the results for the ChIP experiments on brain cells and spleen cells of C57B/6 mice, respectively. Numbers under the graphs indicate the results obtained for the corresponding PCR primer sets shown in panel A.</p

<i>In vivo</i> footprint analysis of the <i>Snrpn</i> promoter region.

<p>LMPCR <i>in vivo</i> footprinting with dimethyl sulfate (DMS) was performed on primary brain cells isolated from Tg^PWS(del) and Tg^AS(del) mice carrying a deletion of the entire murine AS/PWS region on either the paternal or maternal chromosomes, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052390#pone.0052390-Gabriel1" target="_blank">[20]</a>. Cells were treated with increasing time of exposure to DMS. “DNA” represents control samples of purified genomic DNA subjected to DMS treatment and LMPCR in parallel with samples treated <i>in vivo</i> with increasing time of exposure to DMS (60, 90, 120 seconds). The LMPCR primer sets detecting each footprint are described in the text. A) – D) Autoradiograms showing <i>in vivo</i> footprints P1 – P5 in the <i>Snrpn</i> promoter region. Filled circles represent footprints detected as guanines protected from DMS reactivity, open circles represent footprints at guanine sites showing enhanced DMS reactivity. Numbers on the left of each sequencing gel denote the location of each footprint relative to the transcription initiation site. The nucleotide sequences to the right of each autoradiogram show the DNA sequence containing and flanking each footprint; the filled/open circles indicate the location of each <i>in vivo</i> footprint within the DNA sequence and correspond to the footprinted sites shown on the left of each autoradiogram. E) Summary of <i>in vivo</i> footprints in the <i>Snprn</i> promoter region of the paternal allele. The footprinted sites detected by LMPCR <i>in vivo</i> footprinting are shown within the nucleotide sequence of the promoter region of the mouse <i>Snprn</i> gene. Open and filled circles denote footprinted sites as described above. At each footprinted position, the footprinted site corresponds to the strand containing the guanine nucleotide. Shaded nucleotide sequences indicate sequences conserved between the human and mouse <i>Snrpn</i> promoters. Brackets below the nucleotide sequence indicate the location and identity of potential transcription factor binding sites. The bent arrow depicts the transcription initiation site; numbering of nucleotides is relative to the transcription initiation site.</p