Oct4 to Count 2 Anton Wutz1

Oct4 to count 2 Anton Wutz1

1Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria Cell Research (2009) 19:917-919. doi: 10.1038/cr.2009.92; published online 3 August 2009

In mammals, dosage compensation inactivation [3, 4]. The current view is CTCF interacts biochemically with between the sexes involves the tran- that the pairing event is responsible for Oct4, and Sox2 can bind YY1 suggest- scriptional silencing of one of the two X establishing mutual exclusive outcomes ing the formation of a larger complex chromosomes in female cells. Thereby, on the active and the inactive X chro- in ES cells. Importantly, mutation of either the paternally or maternally inher- mosome. Oct4 binding sites in the Tsix promoter ited X chromosome becomes inactivated The molecular basis of pairing has reduces its transcriptional activity in in a random manner. X inactivation is been studied in embryonic stem (ES) a reporter assay. These observations initiated in early embryogenesis. The X cells. Female mouse ES cells possess implicate Oct4 in the regulation of inactivation centre (Xic) regulates the two active X chromosomes, one of Tsix. Oct4 and Sox2 are downregulated initiation of X inactivation in a devel- which becomes inactivated upon dif- in differentiation of ES cells and, thus, opmentally controlled process during ferentiation. Previous work by the Lee could explain the loss of Tsix expression early embryogenesis and contains ele- group has shown that the DNA binding which in turn could enable of Xist ex- ments that signal the presence of an X protein CTCF is essential for Xic pair- pression and lead to the inactivation of chromosome. The Xic also contains the ing, which is mediated through a region the X chromosome. This interpretation non-coding Xist RNA, which localizes close to the Tsix promoter [5]. CTCF is strengthened by the observation that in cis over the X chromosome and trig- binds together with the transcription Oct4 depletion using siRNAs leads to gers chromosome-wide gene silencing. factor YY1 to multiple sites within a reduction of Tsix expression and the The present view is that the mechanisms the Xic, where it might contribute to expression of Xist from both X chro- that control X inactivation converge at Tsix regulation [6]. CTCF is expressed mosomes in differentiated female ES the regulation of Xist. throughout ES cell differentiation and, cells. Thus, in this system Oct4 acts as Xist expression has been studied thus, does not fully explain the devel- a transcriptional activator of Tsix and a in detail. Xist is repressed by the non- opmental onset of X inactivation after repressor of Xist. coding Tsix RNA that overlaps the Xist the onset of differentiation. Yet, a recent The study further examines the role transcription unit in antisense orienta- study links Xist regulation and ES cell of Oct4 for Xic pairing. Depletion of tion [1]. In female cells, Tsix is required transcription factors [7]. Oct4 blocks Xic pairing indicating that to suppress Xist expression from the In their present study, Donohoe and Oct4 also is required for this process. one X chromosome that will remain colleagues examine the function of the The results by Donohoe et al. provide active. In male cells with only one X transcription factors Oct4 and Sox2, an important step forward for under- chromosome Xist remains repressed which show an expression pattern that is standing the developmental regulation if Tsix is disrupted. This suggests that restricted to early ES cell differentiation of X inactivation. The following model the presence of two X chromosomes (for review see [8]), in the developmen- can be proposed: Xic-Xic pairing will predisposes female cells for Xist expres- tal regulation of Xist [9]. Bioinformatic predispose the cells for Xist expression [2]. Before upregulation of Xist the analysis reveals putative Oct4 and Sox2 sion. Yet, at the initiatial stage strong Xic regions on the two X chromosomes binding sites within Xist and at the start Tsix expression will prevent Xist up- pair providing a potential trigger for X site and enhancer of Tsix (Figure 1). regulation. Upon differentiation Oct4 Binding of the factors is confirmed by and Sox2 expression diminishes and chromatin immunoprecipitation. Since leads to a shutdown of Tsix from one Correspondence: Anton Wutz Oct4 binding sites are close to CTCF of the two X chromosomes allowing Tel: +43-1-79730/3430; Fax: +43-1-7987153 binding sites the authors examine a upregulation of Xist and X chromosome E-mail: [email protected] potential interaction. They observe that inactivation. In such a sense Oct4 could npg 918

Xic-Xic This activator remains to be identified. Xic pairing The scenario is further complicated by Xist the fact that a region quite distant from Xist and Tsix has been shown to lead to pairing of the X chromosomes long before initiation of X inactivation [13]. Tsix Xite How this pairing is established and if it is also dependent on Oct4 and CTCF is not known at present. Identification Site of Oct4 binding of Oct4 as a regulator of X inactivation presents a major step forward to address these important questions. Oct4 References

1 Lee JT, Davidow LS, Warshawsky D. Differentiation Tsix, a gene antisense to Xist at the X-inactivation centre. Nat Genet 1999; Figure 1 Summary of Oct4 binding in the Xic. A schematic representation of the 21:400-404. mouse Xic region indicates the non coding Xist and Tsix RNAs. Oct4 binding to 2 Lee JT, Lu N. Targeted mutagenesis of the Tsix promoter and its enhancer Xite activates Tsix, which in turn represses Tsix leads to nonrandom X inactiva- Xist. Down regulation of Oct4 upon differentiation leads to a reduction of Tsix tion. Cell 1999; 99:47-57. transcription. This allows Xist upregulation from one X chromosome when Xic- 3 Bacher CP, Guggiari M, Brors B, et al. Xic pairing triggers X inactivation. Transient colocalization of X-inactivation centres accompanies the initiation of X inactivation. Nat Cell Biol 2006; provide a candidate for a blocking factor quite distant to the Tsix promoter, which 8:293-299. 4 Xu N, Tsai CL, Lee JT. Transient ho- that has been proposed from theoretical is also observed by Donohoe et al. but mologous chromosome pairing marks considerations to explain how one X not further examined. It appears likely the onset of X inactivation. Science chromosome can be kept active. This is that Oct4 contributes to Xist repression 2006; 311:1149-1152. a significant step forward in understand- in Tsix dependent and independent 5 Xu N, Donohoe ME, Silva SS, Lee JT. ing the regulation of X inactivation. modes. Ultimately, deletion of the Oct4 Evidence that homologous X-chro- The results obtained by Donohoe binding site in Xist intron 1 will be re- mosome pairing requires transcription and colleagues also prompt new ques- quired for dissecting its contribution to and Ctcf protein. Nat Genet 2007; 39:1390-1396. tions and will spur further investigation. the regulation of X inactivation. Albeit 6 Donohoe ME, Zhang LF, Xu N, Shi Y, Among the immediate questions is the certain details of the mechanism remain Lee JT. Identification of a Ctcf cofac- function of Oct4 in pluripotent cells that to be elucidated, the data presented by tor, Yy1, for the X chromosome binary have already established X inactiva- Donohoe et al. make a strong case for switch. Mol Cell 2007; 25:43-56. tion. Human embryonic stem cells and Oct4 as an autosomal factor for Xist 7 Navarro P, Chambers I, Karwacki- epiblast derived stem cells from mice regulation. Neisius V, et al. Molecular coupling have already undergone X inactivation Less clear is the X-chromosomal de- of Xist regulation and pluripotency. Science 2008; 321:1693-1695. despite expressing high levels of Oct4 terminant that enables X inactivation in 8 Ying QL, Wray J, Nichols J, et al. The [10, 11]. Explaining Oct4 function in female cells. Presently, data indicate that ground state of embryonic stem cell these different cellular contexts will the pairing of Xic regions could perform self-renewal. Nature 2008; 453:519- require the introduction of additional this function to activate Xist expression. 523. regulatory components. Also the func- However, a recent study has demon- 9 Donohoe ME, Silva SS, Pinter SF, tion of the Oct4 binding site in the first strated that an X chromosome lacking Xu N, Lee JT. The pluripotency fac- intron of Xist is not fully understood. Xic sequences including Tsix can still be tor Oct4 interacts with Ctcf and also This binding site has been recently counted and will trigger X inactivation controls X-chromosome pairing and counting. Nature 2009; 460:128-132. found to exert repression of Xist inde- of the other wild type X chromosome 10 Silva SS, Rowntree RK, Mekhoubad pendent of Tsix transcription [7]. This [12]. This strongly argues for an X- S, Lee JT. X-chromosome inactiva- is entirely consistent with its position linked activator of X-inactivation that tion and epigenetic fluidity in human well within the Tsix transcription unit resides outside the Xist-Tsix region. embryonic stem cells. Proc Natl Acad

Cell Research | Vol 19 No 8 | August 2009 npg 919 Sci USA 2008; 105:4820-4825. 12 Monkhorst K, Jonkers I, Rentmeester 13 Augui S, Filion GJ, Huart S, et al. 11 Guo G, Yang J, Nichols J, et al. Klf4 E, Grosveld F, Gribnau J. X inactiva- Sensing X chromosome pairs before reverts developmentally programmed tion counting and choice is a stochas- X inactivation via a novel X-pairing restriction of ground state pluripo- tic process: evidence for involvement region of the Xic. Science 2007; tency. Development 2009; 136:1063- of an X-linked activator. Cell 2008; 318:1632-1636. 1069. 132:410-421.