Mediator Subunit 12 Is Required for Neutrophil Development in Zebrafish

Hematopoiesis requires the spatiotemporal organization of regulatory factors to successfully orchestrate diverse lineage specificity from stem and progenitor cells. Med12 is a regulatory component of the large Mediator complex that enables contact between the general RNA polymerase II transcriptional machinery and enhancer bound regulatory factors. We have identified a new zebrafish med12 allele, syr, with a single missense mutation causing a valine to aspartic acid change at position 1046. Syr shows defects in hematopoiesis, which predominantly affect the myeloid lineage. Syr has identified a hematopoietic cell-specific requirement for Med12, suggesting a new role for this transcriptional regulator

The Mechanism of Release of P-TEFb and HEXIM1 from the 7SK snRNP by Viral and Cellular Activators Includes a Conformational Change in 7SK

The positive transcription elongation factor, P-TEFb, is required for the production of mRNAs, however the majority of the factor is present in the 7SK snRNP where it is inactivated by HEXIM1. Expression of HIV-1 Tat leads to release of P-TEFb and HEXIM1 from the 7SK snRNP in vivo, but the release mechanisms are unclear.We developed an in vitro P-TEFb release assay in which the 7SK snRNP immunoprecipitated from HeLa cell lysates using antibodies to LARP7 was incubated with potential release factors. We found that P-TEFb was directly released from the 7SK snRNP by HIV-1 Tat or the P-TEFb binding region of the cellular activator Brd4. Glycerol gradient sedimentation analysis was used to demonstrate that the same Brd4 protein transfected into HeLa cells caused the release of P-TEFb and HEXIM1 from the 7SK snRNP in vivo. Although HEXIM1 binds tightly to 7SK RNA in vitro, release of P-TEFb from the 7SK snRNP is accompanied by the loss of HEXIM1. Using a chemical modification method, we determined that concomitant with the release of HEXIM1, 7SK underwent a major conformational change that blocks re-association of HEXIM1.Given that promoter proximally paused polymerases are present on most human genes, understanding how activators recruit P-TEFb to those genes is critical. Our findings reveal that the two tested activators can extract P-TEFb from the 7SK snRNP. Importantly, we found that after P-TEFb is extracted a dramatic conformational change occurred in 7SK concomitant with the ejection of HEXIM1. Based on our findings, we hypothesize that reincorporation of HEXIM1 into the 7SK snRNP is likely the regulated step of reassembly of the 7SK snRNP containing P-TEFb

Proviral HIV-genome-wide and pol-gene specific Zinc Finger Nucleases: Usability for targeted HIV gene therapy

<p>Abstract</p> <p>Background</p> <p>Infection with HIV, which culminates in the establishment of a latent proviral reservoir, presents formidable challenges for ultimate cure. Building on the hypothesis that <it>ex-vivo </it>or even <it>in-vivo </it>abolition <it>or </it>disruption of HIV-gene/genome-action by target mutagenesis or excision can irreversibly abrogate HIV's innate fitness to replicate and survive, we previously identified the isoschizomeric bacteria restriction enzymes (REases) AcsI and ApoI as potent cleavers of the HIV-pol gene (11 and 9 times in HIV-1 and 2, respectively). However, both enzymes, along with others found to cleave across the entire HIV-1 genome, slice (SX) at palindromic sequences that are prevalent within the human genome and thereby pose the risk of host genome toxicity. A long-term goal in the field of R-M enzymatic therapeutics has thus been to generate synthetic restriction endonucleases with longer recognition sites limited in specificity to HIV. We aimed (i) to assemble and construct zinc finger <it>arrays </it>and <it>nucleases </it>(ZFN) with either proviral-HIV-pol gene or proviral-HIV-1 whole-genome specificity respectively, and (ii) to advance a model for pre-clinically testing lentiviral vectors (LV) that deliver and transduce either ZFN genotype.</p> <p>Methods and Results</p> <p><it>First, </it>we computationally generated the consensus sequences of (a) 114 dsDNA-binding zinc finger (Zif) <it>arrays </it>(ZFAs or Zif_HIV-pol) and (b) two zinc-finger <it>nucleases </it>(ZFNs) which, unlike the AcsI and ApoI homeodomains, possess specificity to >18 base-pair sequences uniquely present within the HIV-pol gene (Zif_HIV-polF_N). Another 15 ZFNs targeting >18 bp sequences within the complete HIV-1 proviral genome were constructed (Zif_HIV-1F_N). <it>Second, </it>a model for constructing lentiviral vectors (LVs) that deliver and transduce a diploid copy of either Zif_HIV-polF_Nor Zif_HIV-1F_Nchimeric genes (termed <b>LV- 2xZif</b>_{<b>HIV-pol</b>}<b>F</b>_{<b>N </b>}and <b>LV- 2xZif</b>_<b>HIV-1</b><b>F</b>_{<b>N, </b>}respectively) is proposed. <it>Third, </it>two preclinical models for controlled testing of the safety and efficacy of either of these LVs are described using active HIV-infected TZM-bl reporter cells (HeLa-derived JC53-BL cells) and latent HIV-infected cell lines.</p> <p>Conclusion</p> <p><b>LV-2xZif</b>_{<b>HIV-pol</b>}<b>F</b>_{<b>N </b>}and <b>LV- 2xZif</b>_<b>HIV-1</b><b>F</b>_{<b>N </b>}may offer the <it>ex-vivo </it>or even <it>in-vivo </it>experimental opportunity to halt HIV replication functionally by directly abrogating HIV-pol-gene-action <it>or </it>disrupting/excising over 80% of the proviral HIV DNA from latently infected cells.</p

Mechanisms and models of somatic cell reprogramming

Whitehead Institute for Biomedical Research (Jerome and Florence Brill Graduate Student Fellowship)National Institutes of Health (U.S.) (US NIH grant RO1-CA087869)National Institutes of Health (U.S.) (US NIH grant R37-CA084198)National Science Foundation (U.S.) (NSF Graduate Research Fellowship)National Institutes of Health (U.S.) ((NIH) Kirschstein National Research Service Award,1 F32 GM099153-01A1)Vertex Pharmaceuticals Incorporated (Vertex Scholar

New Insights into the Control of HIV-1 Transcription: When Tat Meets the 7SK snRNP and Super Elongation Complex (SEC)

Recent studies aimed at elucidating the mechanism controlling HIV-1 transcription have led to the identification and characterization of two multi-subunit complexes that both contain P-TEFb, a human transcription elongation factor and co-factor for activation of HIV-1 gene expression by the viral Tat protein. The first complex, termed the 7SK snRNP, acts as a reservoir where active P-TEFb can be withdrawn by Tat to stimulate HIV-1 transcription. The second complex, termed the super elongation complex (SEC), represents the form of P-TEFb delivered by Tat to the paused RNA polymerase II at the viral long terminal repeat during Tat transactivation. Besides P-TEFb, SEC also contains other elongation factors/co-activators, and they cooperatively stimulate HIV-1 transcription. Recent data also indicate SEC as a target for the mixed lineage leukemia (MLL) protein to promote the expression of MLL target genes and leukemogenesis. Given their roles in HIV-1/AIDS and cancer, further characterization of 7SK snRNP and SEC will help develop strategies to suppress aberrant transcriptional elongation caused by uncontrolled P-TEFb activation. As both complexes are also important for normal cellular gene expression, studying their structures and functions will elucidate the mechanisms that control metazoan transcriptional elongation in general

Transcription-independent functions of MYC: regulation of translation and DNA replication

MYC is a potent oncogene that drives unrestrained cell growth and proliferation. Shortly after its discovery as an oncogene, the MYC protein was recognized as a sequence-specific transcription factor. Since that time, MYC oncogene research has focused on the mechanism of MYC-induced transcription and on the identification of MYC transcriptional target genes. Recently, MYC was shown to control protein expression through mRNA translation and to directly regulate DNA replication, thus initiating exciting new areas of oncogene research

Myc-induced proliferation and transformation require Akt-mediated phosphorylation of FoxO proteins

Myc synergizes with Ras and PI3-kinase in cell transformation, yet the molecular basis for this behavior is poorly understood. We now show that Myc recruits TFIIH, P-TEFb and Mediator to the cyclin D2 and other target promoters, while the PI3-kinase pathway controls formation of the preinitiation complex and loading of RNA polymerase II. The PI3-kinase pathway involves Akt-mediated phosphorylation of FoxO transcription factors. In a nonphosphorylated state, FoxO factors inhibit induction of multiple Myc target genes, Myc-induced cell proliferation and transformation by Myc and Ras. Abrogation of FoxO function enables Myc to activate target genes in the absence of PI3-kinase activity and to induce foci formation in primary cells in the absence of oncogenic Ras. We suggest that the cooperativity between Myc and Ras is at least in part due to the fact that Myc and FoxO proteins control distinct steps in the activation of an overlapping set of critical target genes