Topics in Molecular Biology ( ) 3 rd Lecture Hall, Room 504 Thursday 1pm-2:50pm

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1 Topics in Molecular Biology ( ) 3 rd Lecture Hall, Room 504 Thursday 1pm-2:50pm Instructor: Ming Tian Biology Building mingtian@pku.edu.cn Phone: Office hours: Monday 1pm-4pm or by appointment Prerequisite: Molecular Biology Lecture and discussion: Discussion topics will be selected from the areas of gene expression and genomic instability. Lecture and discussion will be based on research and review papers; lecture slides and references will be posted on the teaching website (. Homework, test and grading: Homework will be assigned throughout the semester. There will be a take-home exam at the end of the semester. Final grade will be based on homework (50%) and the takehome exam (50%).

2 Dosage Compensation Female Male Mammal 1 0 Y 1 D. melanogaster 1 1 Y 2 C. elegance inactivation in mammals Two populations of fibroblast clones from a female who is heterozygous for HGPRT (hypoxanthine-guanine phosphoribosyl transferase) mutation. Wild-type HGPRT gene Mutant HGPRT gene Phase contrast 3 H-Guanine incorporation into RNA a i HGPRT + i a HGPRT - 1

3 inactivation is initiated when there are two inactivation centers. The mapping of inactivation center (ic) based on chromosome rearrangements ist is expressed from the ic of the inactive chromosome (i). ic a ist i ist ic a Y ist Control probe for RNA integrity 2

4 ist is expressed from i in mouse. F1 hybrid [Mus musculus domesticus (dom) x Mus spretus (spe)] ist Random -inactivation dom spe dom spe Biased -inactivation T16 dom spe Control ist is a 15kb noncoding RNA. Conservation of repetitive sequences 3

5 ist RNA forms a nuclear foci. 47, cell (two i) ist Fibronectin RNA ist is retained in the nucleus. Visualization of nuclear RNA only 46, cells ist Collagen RNA ist forms a large nuclear foci. Visualization of nuclear RNA only 46, cells ist UBE-1 RNA UBE-1 is expressed from both chromosomes. ist colocalizes with one of the chromosomes. ist RNA paints chromosome. 47, cell chromosome ist RNA 46, cell chromosome ist RNA 46, cell Chromosome 17 Collagen RNA 4

6 The area of ist RNA correlates with that of chromosome. Model of interaction of ist with i. Nuclear matrix The inactive chromosome is associated with heterochromatin markers. H3K9me3 is associated with transcriptional repression in heterochromatin. i is enriched for H3K9me3. i is deficient in H3 and H4 acetylation, which is linked to transcriptional activation. 5

7 Induction of ES cells differentiation in vitro. H3K4me3 is linked to active transcription. H3K4 is hypomethylated on i. Colocalization of H3K27me3 and Ezh2 on i. Ezh2 catalyzes the methylation of H3 on K27. H3K27me3 is linked to transcriptional repression. ist is required for initiating inactivation. F1 129xPGK ES cell (PGK12.1) Differentiated ES clone (EB: embryonic body) 129 PGK Both are active. Differentiation 129 PGK 129 PGK Random inactivation 6

8 Deletion of ist prevents -inactivation. PGK12.1 ES cell 129 PGK T67E1 ES cell 129 m PGK 129 PGK 129 PGK 129 m PGK Random inactivation inactivation on PGK allele ist is not detectable from the targeted 129 allele. ist RT-PCR product Hind3 Hind3 polymorphism 129 allele PGK allele U: uncut C: Hind3 cut Inactivated chromosome is latereplicating. 7

9 ist deletion on 129 allele prevents -inactivation on this allele. Single cell analysis of Pgk-1 expression A: PGK B: 129 Pgk-1 RNA Rps4 RNA Smcx RNA -linked genes Zfx DNA -inactivation pattern changes during mouse development. 8

10 Reactivation of i during germ cell formation. ist RNA is lost from primordial germ cells (PGCs) during female embryonic development. Reactivation of genes on i during germ cell formation Singel cell RT-PCR of transcripts from -linked genes The two alleles are distinguished by polymorphisms. Biallelic expression is marked with *. 9

11 Chromosome Counting 2: transcription of ist on one chromosome. ist a i 1: no ist transcription ist Y inactivation is regulated by an antisense RNA (Tsix) of ist. Use of strand-specific probe in FISH analysis ist sense RNA ist anti-sense RNA 10

12 -inactivation occurs preferentially on the Tsix knockout allele. WT *4xdilution 129 cas 129 cas Tsix knokcout on 129 allele 129 cas ist Tsix Tsix knockout ist RNA Tsix promoter *undifferentiated cell; arrow: transitional cell. Discussion Topic Jonkers, I. et al. RNF12 is an -encoded dose-dependent activator of chromosome inactivation. Cell 139, (2009). Shin, J. et al. Maternal RNF12/RLIM is required for imprinted -chromosome inactivation in mice. Nature 467, (2010). Barakat, T. S. et al. RNF12 activates ist and is essential for chromosome inactivation. PloS Genet. 7, e (2011). 11