Recent Advances in Understanding the Reversal of Gene Silencing During X Chromosome Reactivation

Abstract

Dosage compensation between XX female and XY male cells is achieved by a process known as X chromosome inactivation (XCI) in mammals. XCI is initiated early during development in female cells and is subsequently stably maintained in most somatic cells. Despite its stability, the robust transcriptional silencing of XCI is reversible, in the embryo and also in a number of reprogramming settings. Although XCI has been intensively studied, the dynamics, factors, and mechanisms of X chromosome reactivation (XCR) remain largely unknown. In this review, we discuss how new sequencing technologies and reprogramming approaches have enabled recent advances that revealed the timing of transcriptional activation during XCR. We also discuss the factors and chromatin features that might be important to understand the dynamics and mechanisms of the erasure of transcriptional gene silencing on the inactive X chromosome (Xi).

Keywords: X chromosome inactivation; X chromosome reactivation; chromatin; epigenetic memory; epigenetics; gene silencing; pluripotency; stem cells.

FIGURE 1

XCR during female mouse and human iPSC reprogramming and following cell fusion. Mouse fibroblasts induced to a pluripotent state by the upregulation of Oct4, Sox2, cMyc, and Klf4 activate their Xi. Human fibroblasts converted to the primed pluripotent state by upregulating OCT4, SOX2, cMYC, and KLF4 do not undergo XCR, further reprogramming to the naïve state using a naïve conversion media [e.g., 5iLAF (which contains inhibitors that target the glycogen synthase kinase-3 (GSK3b), the mitogen-activated protein kinase (MEK), the Rho-associated, coiled-coil containing protein kinase (ROCK), the serine/threonine-protein kinase B-Raf (BRAF) and the proto-oncogene tyrosine-protein kinases (SRC), in addition to human leukemia inhibitor factor (LIF), activin A and fibroblast growth factor (FGF) (Theunissen et al., 2014)] or T2iLGö [which requires overexpression of KLF2 and NANOG in the presence of MEK, GSK-3, protein kinase C (PKC), ROCK inhibitor and human LIF (Takashima et al., 2014)] is required to induce XCR. Partial human XCR is also achievable through cell fusion, whereby a human fibroblast is fused to a mouse embryonic stem cell (mESC). Following mitosis, a hybrid cell is formed containing chromosomes from both mouse and human in which the human X chromosome is partially reactivated. Red nuclei = XiXa state, green nuclei = XaXa state, XaXa^∗ = partially reactivated X, XiXe = eroded X.

FIGURE 2

XCI during female mouse and human development. During mouse development, embryonic cells around the 4-cell stage inactivate the paternally inherited X chromosome. Cells of the primitive endoderm and trophectoderm in the preimplantation blastocyst keep this inactivation pattern while those in the epiblast reactivate the paternal Xi. Around implantation, an X chromosome is randomly inactivated within epiblast cells. Following specification of PGCs, these cells activate the Xi. Primary oocytes within the fetus and adult mouse do not contain an Xi while all somatic cells retain the Xi pattern of their epiblast precursor. During human development, random XCI is seen as a gradual process beginning in the early blastocyst and completing just prior to implantation, this pattern of inactivation is retained in all future somatic cells. Following specification of PGCs these cells reactivate the Xi which remains active in all future germ cells. Cells are colored by their lineage displayed in the upper right panel, primary oocytes (green) within the fetus and adults represent the primary oocytes contained in the ovaries. Relative timescales of mouse and human development are not accurately represented here. PGCs, primordial germ cells; Xa, active X chromosome; Xi, inactive X chromosome; Xip, inactive paternally inherited X chromosome. Reproduced and adapted with permission from Pasque and Plath (2015).

FIGURE 3

Stages of XCR. In this model, XCR can be divided into three phases: initiation, progression, and completion of XCR. During the early stages, early genes are transcriptionally activated and Xist repression is initiated. During XCR progression, Xist RNA, H3K27me3, and macroH2A are gradually lost from the Xi. Tsix, mid and late genes are transcriptionally reactivated. This process is coordinated by multiple chromatin pathways (such as UTX) as well as chromatin and transcriptional regulators (HDACs and TFs). In addition, TADs and mega-domains are reacquired throughout the initiation and progression phase of XCR. Finally, completion of XCR is characterized by a complete transcriptional reactivation of silenced genes, DNA hypomethylation, loss of epigenetic memory of XCI (random XCI or imprinted), and competence to initiate random XCI. Xa, active X chromosome; Xi, inactive X chromosome; TADs, topologically associating domains; XCI, X chromosome inactivation.