Download Exercise week 10, with answers File

Exercise for BCM-1
Week 10 (Constam)
November 17, 2014
1) True/false: CpG islands are regions in the genome that are protected
against de novo methylation both in early embryos and in gametes by
specific TFs such as Sp1
True
2) DNase Hypersensitivity Assays are useful
a. to identify relatively open regions in chromatin
b. to map candidate gene regulatory regions
c. to distinguish regions of CpG methylation from CpG islands
d. to determine the DNA footprint of a transcription factor
Both a) and b) are correct. Partial digestion by DNase I is also used in d, but not to
monitor hypersensitivity of specific regions. c) is false because CpG methylation can be
monitored e.g. by methylation-sensitive restriction enzymes, but not by DNase I.
3) True/false: A role for GATA-1 in megakaryocytes has been confirmed by
a. the accumulation of undifferentiated, rapidly proliferating
megakaryocyte/erythrocyte progenitors in a subset of leukemia patients
b. the lack of blood platelets in female mice lacking one copy of Gata1
c. targeted deletion of a hematopoietic cell-specific enhancer in the Gata1
regulatory region
d. promoter bashing studies showing that a fragment of the Gata1
regulatory region can direct lacZ expression in megakaryocytes
b) is false: Female Gata1+/- mice still develop normal platelets, even though
the single wild-type copy of Gata1 is silenced by X-chromosome inactivation
in 50% of their cells.
d) is not exactly true either: 'Promoter bashing' (d) is a method to test the
potential of candidate gene regulatory regions to drive the expression of a
reporter in specific tissues. This approach cannot directly prove a function for
Gata1 in these cells, although it may provide indirect supporting evidence
4) Feed-forward loops in gene regulatory networks
a. describe the behavior e.g. of AP-1 complexes or myogenic TFs such as
MyoD to amplify their own expression
b. accelerate the induction of target genes
c. delay the induction of target genes and filter specific signals from
randomly fluctuating inputs ('noise')
d. typically act on super-enhancers
Only c is correct. Self amplification loops (positive feedback) where A
induces A is common among these and many other TFs, but should be
distinguished from feedforward loops where A induces B to induce C. A
feedforward loop will be blocked by cycloheximide, whereas positive
feedback is not. Super-enhancers boost autoinduction, not feedforward
loops.
5) In mammalian embryos, Sox2 and the POU domain factor Oct4 are
required at the blastocyst stage to specify the pluripotent inner cell mass
(ICM) cells that give rise to all cells and tissues of the future body. Forced
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expression of these 2 factors together with c-Myc and Klf4 (a Krüppel-like
zinc finger TF) in differentiated cells is sufficient to induce their
"reprogramming". Which one of the following statements is not entirely
correct:
a. Reprogramming describes a process of induced cell de-differentiation to
a pluripotent state
b. All of these reprogramming factors ('KSOM') activate genes that control
specific developmental processes and stem cell self-renewal
c. Multiple reprogramming factors synergize by simultaneously binding to
so-called super-enhancers
d. ES cell-specific super-enhancers facilitate the recruitment of such
pioneer factors to closed chromatin
e. Reprogramming factors recruit Tet enzymes to convert 5-methyl
cytosines to 5-hydroxymethyl cytosine and thereby trigger CpG
demethylation
b) is only half true: We have seen that c-Myc instead is known to bind to
thousands of genes that generally regulate cell proliferation, RNA processing
and metabolism. Interestingly, among the 4 'Yamanaka factors' KSOM, cMyc is the only one that is not absolutely required for reprogramming. It only
increases the efficiency of the process.
6) R26RLSL reporter mice carry the stop-floxed lacZ cassette in the
ubiquitously expressed Rosa26 locus. The R26RLSL reporter has been
engineered for genetic lineage tracing, e.g. to trace the fate of MyoDexpressing myoblasts. If you cross R26RLSL reporter into Sox2+/Cre mice
which express Cre recombinase from the Sox2 locus, which cells and tissues
will switch on the lacZ reporter allele?
Since Sox2 is transiently expressed at the blastocyst stage in the pluripotent
ICM cells that will go on to form all tissues of the future embryos, Cre is
active and deletes the stop-flox cassette of the R26RLSL reporter at that
stage. Henceforth, lacZ will remain permanently expressed under the control
of the Rosa26 promoter and in all ICM-descendant cells, i.e. in all tissues of
the body, even when they no longer express Sox2.
7) True/false: Dosage compensation of sex chromosomes in mammals is
achieved by
a. by inactivating one X-chromosome of female cells in so-called 'Barr
bodies'
b. by transcribing the long non-coding Xist RNA from the XIC region on the
X-chromosome
c. by recruiting a repressive histone (macroH2A) and repressive histone
methyl-transferases to the inactive X
d. through widespread de novo DNA methylation
All of these statements are true
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