Basal transcription of the HIV LTR is highly repressed and requires Tat to recruit the positive transcription elongation factor, P-TEFb, which functions to promote the transition of RNA polymerase II from abortive to productive elongation. P-TEFb is found in two forms in cells, a free, active form and a large, inactive complex that also contains 7SK RNA and HEXIM1 or HEXIM2. Here we show that HIV infection of cells led to the release of P-TEFb from the large form. Consistent with Tat being the cause of this effect, transfection of a FLAG-tagged Tat in 293T cells caused a dramatic shift of P-TEFb out of the large form to a smaller form containing Tat. In vitro, Tat competed with HEXIM1 for binding to 7SK, blocked the formation of the P-TEFb–HEXIM1–7SK complex, and caused the release P-TEFb from a pre-formed P-TEFb–HEXIM1–7SK complex. These findings indicate that Tat can acquire P-TEFb from the large form. In addition, we found that HEXIM1 binds tightly to the HIV 5′ UTR containing TAR and recruits and inhibits P-TEFb activity. This suggests that in the absence of Tat, HEXIM1 may bind to TAR and repress transcription elongation of the HIV LTR.

Background: The positive transcription elongation factor, P-TEFb, comprised of cyclin dependent kinase 9 (Cdk9) and cyclin T1, T2 or K regulates the productive elongation phase of RNA polymerase II (Pol II) dependent transcription of cellular and integrated viral genes. P-TEFb containing cyclin T1 is recruited to the HIV long terminal repeat (LTR) by binding to HIV Tat which in turn binds to the nascent HIV transcript. Within the cell, P-TEFb exists as a kinase-active, free form and a larger, kinase-inactive form that is believed to serve as a reservoir for the smaller form.

Landmark cancer genome resequencing efforts are leading to the identification of mutated genes in many types of cancer. The extreme diversity of mutations being detected presents significant challenges to subdivide causal from coincidental mutations to elucidate how disrupted regulatory networks drive cancer processes. Given that a common early perturbation in solid tumor initiation is bypass of matrix-dependent proliferation restraints, we sought to functionally interrogate colorectal cancer candidate genes (CAN-genes) to identify driver tumor suppressors. We have employed an isogenic human colonic epithelial cell (HCEC) model to identify suppressors of anchorage-independent growth by conducting a soft agar-based short hairpin RNA (shRNA) screen within the cohort of CAN-genes. Remarkably, depletion of 65 of the 151 CAN-genes tested collaborated with ectopic expression of K-RAS V12 and/or TP53 knockdown to promote anchorage-independent proliferation of HCECs. In contrast, only 5 of 362 random shRNAs (1.4%) enhanced soft agar growth. We have identified additional members of an extensive gene network specifying matrix-dependent proliferation, by constructing an interaction map of these confirmed progression suppressors with approximately 700 mutated genes that were excluded from CAN-genes, and experimentally verifying soft agar growth enhancement in response to depletion of a subset of these genes. Collectively, this study revealed a profound diversity of nodes within a fundamental tumor suppressor network that are susceptible to perturbation leading to enhanced cell-autonomous anchorage-independent proliferative fitness. Tumor suppressor network fragility as a paradigm within this and other regulatory systems perturbed in cancer could, in large part, account for the heterogeneity of somatic mutations detected in tumors. Cancer Res; 71(13); 4359-65. Ó2011 AACR.

Background: The TNF gene is regulated in a cell type-specific fashion.Results: A TNF locus site that interacts with the nuclear matrix and proteins that affect DNA torsional stress is selectively accessible in monocytic cells.Conclusion: Monocyte-specific accessibility of the site provides a mechanism of cell type-specific TNF gene regulation.Significance: Cell type-specific chromatin organization elucidates control of TNF regulation and dysregulation.