The onset of random X chromosome inactivation in mouse requires the switch from a symmetric to an asymmetric state, where the identities of the future inactive and active X chromosomes are assigned. This process is known as X chromosome choice. Here, we show that RIF1 and KAP1 are two fundamental factors for the definition of this transcriptional asymmetry. We found that at the onset of differentiation of mouse embryonic stem cells (mESCs), biallelic up-regulation of the long non-coding RNA Tsix weakens the symmetric association of RIF1 with the Xist promoter. The Xist allele maintaining the association with RIF1 goes on to upregulate Xist RNA expression in a RIF1-dependent manner. Conversely, the promoter that loses RIF1 gains binding of KAP1, and KAP1 is required for the increase in Tsix levels preceding the choice. We propose that the mutual exclusion of Tsix and RIF1, and of RIF1 and KAP1, at the Xist promoters establish a self-sustaining loop that transforms an initially stochastic event into a stably inherited asymmetric X-chromosome state.

The glucocorticoid (GR) and androgen (AR) receptors execute unique functions in vivo, yet have nearly identical DNA binding specificities. To identify mechanisms that facilitate functional diversification among these transcription factor paralogs, we studied them in an equivalent cellular context. Analysis of chromatin and sequence suggest that divergent binding, and corresponding gene regulation, are driven by different abilities of AR and GR to interact with relatively inaccessible chromatin. Divergent genomic binding patterns can also be the result of subtle differences in DNA binding preference between AR and GR. Furthermore, the sequence composition of large regions (>10 kb) surrounding selectively occupied binding sites differs significantly, indicating a role for the sequence environment in guiding AR and GR to distinct binding sites. The comparison of binding sites that are shared shows that the specificity paradox can also be resolved by differences in the events that occur downstream of receptor binding. Specifically, shared binding sites display receptor-specific enhancer activity, cofactor recruitment and changes in histone modifications. Genomic deletion of shared binding sites demonstrates their contribution to directing receptor-specific gene regulation. Together, these data suggest that differences in genomic occupancy as well as divergence in the events that occur downstream of receptor binding direct functional diversification among transcription factor paralogs.

The glucocorticoid (GR) and androgen (AR) receptors execute unique functions in vivo, yet have nearly identical DNA binding specificities. To identify mechanisms that facilitate functional diversification among these transcription factor paralogs, we studied AR and GR in an equivalent cellular context. Analysis of chromatin and sequence features suggest that divergent binding, and corresponding gene regulation, are driven by different abilities of AR and GR to interact with relatively inaccessible chromatin. Divergent genomic binding patterns can also be the results of subtle differences in DNA binding preference between AR and GR. Furthermore, the sequence composition of large regions (>10 kb) surrounding selectively occupied binding sites differs significantly, indicating a role for the sequence environment in selectively guiding AR and GR to distinct binding sites. The comparison of binding sites that are shared between AR and GR shows that the specificity paradox can also be resolved by differences in the events that occur downstream of receptor binding. Specifically, we find that shared binding sites display receptor-specific enhancer activity, cofactor recruitment and changes in histone modifications. Genomic deletion of shared binding sites demonstrates their contribution to directing receptor-specific gene regulation. Together, these data suggest that differences in genomic occupancy as well as divergence in the events that occur downstream of receptor binding direct functional diversification among transcription factor paralogs.

Protein-protein interactions (PPIs) play a crucial role in numerous molecular processes. Despite many efforts, mechanisms governing molecular recognition between interacting proteins remain poorly understood and it is particularly challenging to predict from sequence whether two proteins can interact. Here we present a new method to tackle this challenge using intrinsically disordered regions (IDRs). IDRs are protein segments that are functional despite lacking a single invariant threedimensional structure. The prevalence of IDRs in eukaryotic proteins suggests that

Dosage compensation for the X chromosome-linked genes in female placental mammals is achieved through the random silencing of one of the two X chromosomes.The onset of random X inactivation in mouse embryos and in differentiating embryonic stem cells requires the switch from a symmetric state, where both X chromosomes are equivalent, to an asymmetric state, where the identity of the future inactive and active X chromosomes are assigned. This "choice", initiated by a stochastic event, needs to evolve into a stable and transmissible state. The transition from bi-to mono-allelic expression of the long non-coding RNA Tsix is thought to be one of the initial events breaking the symmetry of the two X chromosomes. Here we show that the asymmetric expression of Tsix triggers in turn the switch of RIF1 association with the Xist promoter from dynamic and symmetric to stable and asymmetric (on the future inactive X). On the future inactive X, RIF1 then plays an essential role in the upregulation of Xist, thus initiating the consolidation and stable transmission of the identity of the inactive X.Tsix-dependent exclusion of RIF1 from the future active X chromosome in turn permits the association of KAP1 with the Xist promoter, thus marking the future active X chromosome. Timely mono-allelic association of KAP1 is important for a stable choice and for X inactivation. We present here a double-bookmarking system, based on the mutually exclusive relationships of Tsix and RIF1, and RIF1 and KAP1. This system coordinates the identification of the active and inactive X chromosomes and initiates a self-sustaining loop that transforms an initially stochastic event into a stably inherited asymmetric X chromosome state.