Role of Bright/ARID3A in mouse development, somatic cell reprogramming, and pluripotency

textBright/ARID3A was initially discovered for its role in immunoglobulin heavy chain transcription in the mouse. Bright has also been implicated as a target of p53 and as an E2F binding partner. We have previously shown that Bright is necessary for hematopoietic stem cell development in the embryo. In this work, we show that Bright has a much broader role in development than previously appreciated. Loss of Bright in mice usually results in embryonic lethality due to lack of hematopoietic stem cells. Rare survivor mice initially appear smaller in size than either wildtype or heterozygous littermates, but as they age, these differences diminish. We show that adult Bright null mice have age-dependent kidney defects. Previous work in the adult mouse has not indicated a role for Bright in kidney function. We observed an increase in cellular proliferation in Bright null kidneys, indicating a possible mechanism behind our observation. Loss of Bright has recently been implicated in causing developmental plasticity in somatic cells. Our data indicate that loss of Bright is sufficient to fully reprogram mouse embryonic fibroblasts (MEFs) back to a pluripotent state. We term these cells Bright repression induced pluripotent stem cells (BriPS). BriPS derived from Bright knockout MEFs can be stably maintained in standard embryonic stem cell culture conditions: they express pluripotency markers and can form teratomas in vivo. We further viii show that Bright is active during embryonic stem cell differentiation. Bright represses key pluripotency genes, suggesting the mechanism of reprogramming may be Bright’s direct repression of key pluripotency factors in somatic cells. Recent advances in inducing pluripotency in somatic cells (iPS cells) have created a new field of disease modeling, increased our knowledge of how pluripotency is regulated, and introduced the hope that they can be adapted to treat disease. However, current methods for producing iPS involve overexpression of potentially oncogenic transcription factors, leaving a large gap between the lab and the clinic. Our results mark the first demonstration of an alternative method to reprograming somatic cells that is not mediated by overexpression of pluripotency factors.Cellular and Molecular Biolog

Arid3a regulates mesoderm differentiation in mouse embryonic stem cells

Research into regulation of the differentiation of stem cells is critical to understanding early developmental decisions and later development growth. The transcription factor ARID3A previously was shown to be critical for trophectoderm and hematopoetic development. Expression of ARID3A increases during embryonic differentiation, but the underlying reason remained unclear. Here we show that Arid3a null embryonic stem (ES) cells maintain an undifferentiated gene expression pattern and form teratomas in immune-compromised mice. However, Arid3a null ES cells differentiated in vitro into embryoid bodies (EBs) significantly faster than control ES cells, and the majority forming large cystic embryoid EBs. Analysis of gene expression during this transition indicated that Arid3a nulls differentiated spontaneously into mesoderm and neuroectoderm lineages. While young ARID3A-deficient mice showed no gross tissue morphology, proliferative and structural abnormalities were observed in the kidneys of older null mice. Together these data suggest that ARID3A is not only required hematopoiesis, but is critical for early mesoderm differentiation

Discovery of Novel Isoforms of Huntingtin Reveals a New Hominid-Specific Exon

<div><p>Huntington’s disease (HD) is a devastating neurological disorder that is caused by an expansion of the poly-Q tract in exon 1 of the Huntingtin gene (HTT). HTT is an evolutionarily conserved and ubiquitously expressed protein that has been linked to a variety of functions including transcriptional regulation, mitochondrial function, and vesicle transport. This large protein has numerous caspase and calpain cleavage sites and can be decorated with several post-translational modifications such as phosphorylations, acetylations, sumoylations, and palmitoylations. However, the exact function of HTT and the role played by its modifications in the cell are still not well understood. Scrutiny of HTT function has been focused on a single, full length mRNA. In this study, we report the discovery of 5 novel <i>HTT</i> mRNA splice isoforms that are expressed in normal and <i>HTT</i>-expanded human embryonic stem cell (hESC) lines as well as in cortical neurons differentiated from hESCs. Interestingly, none of the novel isoforms generates a truncated protein. Instead, 4 of the 5 new isoforms specifically eliminate domains and modifications to generate smaller HTT proteins. The fifth novel isoform incorporates a previously unreported additional exon, dubbed 41b, which is hominid-specific and introduces a potential phosphorylation site in the protein. The discovery of this hominid-specific isoform may shed light on human-specific pathogenic mechanisms of HTT, which could not be investigated with current mouse models of the disease.</p></div

Identification of a novel HTT isoform incorporating a previously unreported hominid-specific exon.

<p>(A) RNAseq analysis showing the clear incorporation of a non-reported exon. (B) Alignment of the genomic sequences of exon 41b of mouse and primates, demonstrating the very recent acquisition of this exon along human evolution. Only hominidae family members (in red) have both splice donor and acceptor and maintain the frame. (C) RT-PCR with primers specific for HTT-41b isoform unmistakably detects expression of HTT-41b in hES cells, without amplifying the canonical HTT isoform. (D) Quantification of HTT-41b isoform expression through qRT-PCR at different time points upon neural differentiation of hES cells. Results are shown as mean+SEM of 3–6 independent replicates.</p

PCR and qPCR validation of novel HTT isoforms.

<p>(A) RT-PCR results with primers specific for each individual splice isoform, detecting expression of HTT-Δ10, Δ12, Δ13 and Δ46 in hESCs cells. A plasmid containing the canonical full-length HTT was used to control for non-specific amplification of canonical HTT mRNA. (B) Quantification of HTT isoform expression in hES cells through qPCR in GENEA019 and GENEA020 hESCs. Data represents mean + SEM of 3 replicates.</p

Time course of <i>HTT</i> isoform expression in hESCs differentiating to telencephalic neural fate.

<p>(A-D) RUES2 hESCs were differentiated to neural fate by blocking both branches of TGFβ signaling (default mechanism) as described in Materials and Methods. Values are normalized by GAPDH and displayed as fold change to day 0 values. Only the HTT-Δ10 isoform consistently decreases as the cells differentiate, while all three other isoforms maintain their expression levels unchanged. Error bars represent the standard error of the mean of 3 to 6 independent replicates. * p<0.05 vs d0.</p

RNA-seq analysis reveals novel isoforms of <i>HTT</i>.

<p>Using Tuxedo software, 10 putative <i>HTT</i> splice isoforms were detected in RNA-seq data. (A) RUES2 RNA-seq reads were aligned to the hg19 genome, demonstrating good coverage of HTT mRNA used for the isoform analysis. (B) Diagram depicting the 5 HTT isoforms validated in this study. (C) Frequency of all detected isoforms across the three RNA-seq samples. aSA: alternative splice acceptor, aSD: alternative splice donor.</p

HTT protein consequences of the 4 novel shorter isoforms.

<p>(A, C-E) Alignment of sequencing data obtained from amplicons shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127687#pone.0127687.g002" target="_blank">Fig 2</a> to canonical HTT, confirming the isoform sequences obtained from the RNAseq analysis. Red protein sequences represent predicted lost amino acids. Amino acids highlighted with an asterisk represent Ser434 in (A) and Asp552 in (C), sites of phosphorylation and caspase cleavage, respectively. (B) Alignment of human, mouse, frog and zebrafish HTT mRNA sequences show that Exon10 is specifically missing in the gene model of frog Htt, while it is present in mammalians (human, mouse) and fish (zebrafish).</p

Bright/Arid3A Acts as a Barrier to Somatic Cell Reprogramming through Direct Regulation of Oct4, Sox2, and Nanog

We show here that singular loss of the Bright/Arid3A transcription factor leads to reprograming of mouse embryonic fibroblasts (MEFs) and enhancement of standard four-factor (4F) reprogramming. Bright-deficient MEFs bypass senescence and, under standard embryonic stem cell (ESC) culture conditions, spontaneously form clones that in vitro express pluripotency markers, differentiate to all germ lineages, and in vivo form teratomas and chimeric mice. We demonstrate that BRIGHT binds directly to the promoter/enhancer regions of Oct4, Sox2, and Nanog to contribute to their repression in both MEFs and ESCs. Thus, elimination of the BRIGHT barrier may provide an approach for somatic cell reprogramming