TEST 2 Practice 1. Each abstract describes a particular enhanceosome complex that activates or represses transcription of a gene. Sketch a labeled diagram of the pathway, including as many protein/complex interactions as possible. Explain how the complex works, based on what we learned in class about the fundamental mechanisms of eukaryotic gene regulation. You may use the Internet to look up information. Abstract A The highly conserved zinc-finger protein, CTCF, is a candidate tumor suppressor protein that binds to highly divergent DNA sequences. CTCF has been connected to multiple functions in chromatin organization and gene regulation including chromatin insulator activity and transcriptional enhancement and silencing. Here we show that CTCF harbors several autonomous repression domains. One of these domains, the zinc-finger cluster, silences transcription in all cell types tested and binds directly to the co-repressor SIN3A. Two distinct regions of SIN3A, the PAH3 domain and the extreme C-terminal region, bind independently to this zinc-finger cluster. Analysis of nuclear extract from HeLa cells revealed that CTCF is also capable of retaining functional histone deacetylase activity. Furthermore, the ability of regions of CTCF to retain deacetylase activity correlates with the ability to bind to SIN3A and to repress gene activity. We suggest that CTCF driven repression is mediated in part by the recruitment of histone deacetylase activity by SIN3A. Abstract B We report the cDNA cloning of SREBP-2, the second member of a family of basic-helix-loop-helix-leucine zipper (bHLH-Zip) transcription factors that recognize sterol regulatory element 1 (SRE-1). SRE-1, a conditional enhancer in the promoters for the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A synthase genes, increases transcription in the absence of sterols and is inactivated when sterols accumulate. Human SREBP-2 contains 1141 amino acids and is 47% identical to human SREBP-1a, the first recognized member of this family. The resemblance includes an acidic NH2 terminus, a highly conserved bHLH-Zip motif (71% identical), and an unusually long extension of 740 amino acids on the COOH-terminal side of the bHLH-Zip region. SREBP-2 possesses one feature lacking in SREBP-1a-namely, a glutamine-rich region (27% glutamine over 121 residues). In vitro SREBP-2 bound SRE-1 with the same specificity as SREBP-1a. In vivo it mimicked SREBP-1a in activating transcription of reporter genes containing SRE-1. As with SREBP-1a, activation by SREBP-2 occurred in the absence and presence of sterols, abolishing regulation. Cotransfection of low amounts of pSREBP-1a and pSREBP-2 into human embryonic kidney 293 cells stimulated transcription of promoters containing SRE-1 in an additive fashion. At high levels transcription reached a maximum, and the effects were no longer additive. The reason for the existence of two SREBPs and the mechanism by which they are regulated by sterols remain to be determined. Abstract C. The Notch signaling pathway appears to perform an important function in a wide variety of organisms and cell types. In our present study, we provide evidence that UV irradiation-induced Tip60 proteins reduced Notch1 activity to a marked degree. Accumulated UV irradiation-induced Tip60 suppresses Notch1 transcriptional activity via the dissociation of the Notch1-IC-CSL complex. The binding between endogenous Tip60 and Notch1-IC in UV radiation-exposed cells was verified in this study by coimmunoprecipitation. Interestingly, the physical interaction of Tip60 with Notch1-IC occurs to a more profound degree in the presence of CSL but does not exist in a trimeric complex. Using Notch1-IC and Tip60 deletion mutants, we also determined that the N terminus, which harbors the RAM domain and seven ankyrin repeats of Notch1-IC, interacts with the zinc finger and acetyl coenzyme A domains of Tip60. Furthermore, here we report that Notch1-IC is a direct target of the acetyltransferase activity of Tip60. Collectively, our data suggest that Tip60 is an inhibitor of the Notch1 signaling pathway and that Tip60-dependent acetylation of Notch1-IC may be relevant to the mechanism by which Tip60 suppresses Notch1 signaling. Abstract D. HATs (histone acetyltransferases) contribute to the regulation of gene expression, and loss or dysregulation of these activities may link to tumorigenesis. Here, we demonstrate that expression levels of HATs, p300 and CBP [CREB (cAMP-response-element-binding protein)-binding protein] were decreased during chemical hepatocarcinogenesis, whereas expression of MOZ (monocytic leukaemia zinc-finger protein; MYST3)--a member of the MYST [MOZ, Ybf2/Sas3, Sas2 and TIP60 (Tat-interacting protein, 60 kDa)] acetyltransferase family--was induced. Although the MOZ gene frequently is rearranged in leukaemia, we were unable to detect MOZ rearrangement in livers with hyperplastic nodules. We examined the effect of MOZ on hepatocarcinogenic-specific gene expression. GSTP (glutathione S-transferase placental form) is a Phase II detoxification enzyme and a well-known tumour marker that is specifically elevated during hepatocarcinogenesis. GSTP gene activation is regulated mainly by the GPE1 (GSTP enhancer 1) enhancer element, which is recognized by the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2)-MafK heterodimer. We found that MOZ enhances GSTP promoter activity through GPE1 and acts as a co-activator of the Nrf2-MafK heterodimer. Further, exogenous MOZ induced GSTP expression in rat hepatoma H4IIE cells. These results suggest that during early hepatocarcinogenesis, aberrantly expressed MOZ may induce GSTP expression through the Nrf2-mediated pathway. Abstract E. A series of transcription
factors critical for maintenance of the neural stem cell state have been
identified, but the role of functionally important corepressors in maintenance
of the neural stem cell state and early neurogenesis remains unclear.
Previous studies have characterized the expression of both SMRT (also
known as NCoR2, nuclear receptor co-repressor 2) and NCoR in a variety
of developmental systems; however, the specific role of the SMRT corepressor
in neurogenesis is still to be determined. Here we report a critical role
for SMRT in forebrain development and in maintenance of the neural stem
cell state. Analysis of a series of markers in SMRT-gene-deleted mice
revealed the functional requirement of SMRT in the actions of both retinoic-acid-dependent
and Notch-dependent forebrain development. In isolated cortical progenitor
cells, SMRT was critical for preventing retinoic-acid-receptor-dependent
induction of differentiation along a neuronal pathway in the absence of
any ligand. Our data reveal that SMRT represses expression of the jumonji-domain
containing gene JMJD3, a direct retinoic-acid-receptor target that functions
as a histone H3 trimethyl K27 demethylase and which is capable of activating
specific components of the neurogenic program. 2. Explain the various classes of repetitive DNA in the human genome. Explain which classes are related to viruses (or not); which classes may still replicate or transpose (or not); and cite examples of possible genetic or medical consequences of specific repetitive sequences. If you use Google, include the source URL. 3. Outline three different kinds of statistical analysis
of a microarray experiment (assuming a good number of replicates.) Explain
what is learned from each, as well as the limitations of each analysis. 4. Identify this type of transcription
factor domain. Explain its molecular structure,
including how amino acids hold it together, and how it binds DNA. Explain
its most likely biological function. 5. Explain the type of RNA helix packing shown in this RNA-protein complex. Also explain the precise peptide-RNA contacts that hold the complex together. 6. Explain how the process of translation balances the need for speed and accuracy of peptide synthesis. Explain why energy is needed for translation, and explain how the expenditure of energy is coupled to the translation cycle. 7. Explain how one figure of Iborra et al shows evidence of translation occurring in the nucleus. Suggest a possible critique of the experiment; how might a researcher argue against the data of this figure. 8. Explain the molecular basis of the herpes virus replication cycle, and how it differs from cellular reproduction. 9. Explain the molecular process of the splicing reaction. Explain how specific experiments led to specific discoveries about splicing.
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