Assistant Professor, Neurology and Neuroscience
Assistant Professor, Cell and Developmental Biology
Weill Cornell Medical College
The goal of the Donohoe laboratory is to investigate the mechanisms of allelic choice and cell fate decisions in development and disease. We have several ongoing projects on epigenetics in stem cells and early development in addition to disease models.
A crucial epigenetic event early in development is dosage compensation. Dosage compensation in mammals is accomplished by silencing one of two female X chromosomes to ensure equal gene expression between XX females and XY males. In mice, this silencing emanates from the X-chromosome inactivation center (Xic) and is controlled in cis by non-coding RNAs: Xist (inactive X-specific transcript); its anti-sense partner, Tsix; and the enhancer-bearing Xite. Xite and Tsix together facilitate “counting” of X chromosomes and the designation of the active X. Xist and Tsix are expressed on both X chromosomes in undifferentiated female mouse embryonic stem cells, an ex vivo system that faithfully recapitulates X-chromosome inactivation (XCI) in the embryo. Upon differentiation, Xite and Tsix are repressed; Xist RNA accumulates and recruits a battery of co-repressors along the inactive X chromosome to initiate silencing. Conversely, Xite and Tsix expression block Xist RNA from silencing designating the future active chromosome. Genetic studies show that Xite and Tsix regulate Xist in cis. However, to ensure the mutually exclusive designation of an active and inactive X, the initiation of XCI requires a trans communication between the two female X chromosomes. Consistent with this hypothesis, we determined that the two female X chromosomes transiently touch prior to XCI and is mediated by Xite and Tsix. X-X kissing correlates with ES cell differentiation. Failure to pair Xs blocks XCI. Autosomal (A) integration of Xite or Tsix DNA sequences induces X-A pairing. This X-A interaction interferes with XCI in female cells. We determined that sub fragments of Xite or Tsix could recapitulate pairing. A common denominator is the presence of the insulator protein, Ctcf, essential for X-X pairing.
Several long, non-coding RNAs control XCI in cis: the silencer Xist; and its two repressors Xite and Tsix. Together Xite and Tsix facilitate “counting” of X chromosomes (XCI occurs when there is more than one X) and the “choice” of the active versus inactive X-chromosome. Previously we determined that the chromatin insulator Ctcf and its co-factor Yy1 regulate Tsix, providing an epigenetic switch for XCI. Ctcf plays an additional role, as its presence is required within Xite and Tsix for the trans communication between the two X chromosomes, an event critical to ensure the mutually exclusive designation of the active and inactive X chromosome. Xite trans-factors Ctcf and Yy1 (Donohoe, et al Mol Cell, 2007; Donohoe, et al Nature, 2009) and Tsix expression are associated with the pluripotent state and X-X homologous pairing correlates with embryonic stem (ES) cell differentiation. Our findings show that Ctcf partners with the pluripotent factor Oct4 in undifferentiated ES cells consistent with the hypothesis that Ctcf acts combinatorially in an early complex. Oct4 regulates XCI by triggering X chromosome interaction and counting. These studies have unraveled a transcriptional circuitry for XCI, linking this epigenetic process with ES cell differentiation. We continue our studies of transcriptional circuitry in XCI, in development, and in disease models.
Dr. Donohoe trained with Dr. Jeannie Lee and Dr. Yang Shi at Massachusetts General Hospital and Harvard Medical School in Boston. Her expertise is in gene regulation and epigenetics in development.
Kamikawa Y, Donohoe ME (2013). The dynamics of X-chromosome inactivation in mouse development. Mol Reprod Dev doi: 10.1002/mrd.22282.
Donohoe ME (2012). Balancing the dose in the mouse. In: JZ Kubiak (Ed.) Mouse Development, Results Problems in Cell Differentiation. Springer; 55:231-45.
Donohoe ME, Silva S, Pinter S, Xu N, Lee JT (2009). The pluripotency factor Oct4 interacts with Ctcf and also control X-chromosome pairing and counting. Nature 460(7251):128-132.
Sleiman SF, Basso M, Mahishi L, Kozikowski AP, Donohoe ME, Langley B, Ratan RR (2009). Putting the 'HAT' back on survival signalling: the promises and challenges of HDAC inhibition in the treatment of neurological conditions. Expert Opin Investig Drugs 18(5):573-84.
Lindroth AM, Park YJ, McLean CM, Dokshin GA, Bernstein JM, Herman H, Pasisni D, Miro X, Donohoe ME, Lee JT, Helin K, Soloway PD (2008). Antagonism between DNA and H3K27methylation at the Imprinted Rasgrf1 Locus. PLOS Genetics 4(8):e1000145.
Xu N*, Donohoe ME* and Lee JT (2007). Evidence that homologous X-chromosome pairing requires transcription and Ctcf protein. *equal contribution. Nat Genet. 2007 Nov; 39(11):1390-6. Epub 2007 Oct 21.
Donohoe ME, Zhang L-F, Xu N, Shi Y, and Lee JT (2007). Identification of a Ctcf Co-factor, Yy1, for the X-chromosome Binary Switch. Mol Cell. 25: 43-56.
Ctcf Interacting Proteins in X-chromosome Inactivation and Development
The pluripotency of embryonic stem (ES) cells is mediated by defined transcription factors. The differentiation of mouse ES cells undergoing global epigenetic reprogramming is exemplified by X-chromosome inactivation (XCI) in which one female X chromosome is silenced to ensure equal gene dosage between male (XY) and female (XX). Somatic XCI is regulated by homologous X-chromosome pairing and counting and by the random choice of future active and inactive X chromosomes. XCI and cell differentiation are tightly coupled as blocking one process, compromises the other and dedifferentiation of somatic cells to induced pluripotent stem cells (iPS) is accompanied by X chromosome reactivation. At the onset of XCI, the two female X-chromosomes transiently pair. This trans-communication is mediated by a 15-kb region within the X-chromosome inactivation center (Xic) at Xite and Tsix, two regulatory non-coding RNAs. (Xu et al, Science, 2006). We have demonstrated that the pluripotency factor Oct4 directly binds Tsix and Xite and also complexes with the trans-factors Ctcf and Yyl (Donohoe, et al, Nature, 2009). Depletion of Oct4 blocks homologous X-X pairing and results in the inactivation of both X chromosomes in female cells. Oct4 is the first trans-factor that regulates X chromosome counting. We continue our search for Ctcf and Oct4 interacting proteins in XCI and early development.
Allelic Choice in Rett Syndrome
Rett Syndrome (RTT) is a neurodevelopmental disorder that is one of the leading causes of mental retardation and autistic behavior in girls, affecting 1/10,000 females. RTT is caused by mutations in the X-linked Methyl CpG-binding protein 2 (MeCP2) gene, which accounts for approximately 80% of sporadic and 45% of familial RTT cases. Phenotypic variation ranging from mild to severe manifestations is observed in RTT. A major cause of this clinical variability is the pattern of X-chromosome inactivation (XCI), a crucial epigenetic process that randomly silences one of the two female X chromosomes in the soma to balance the gene dosage with XY males. Favorable XCI that preferentially silences the X chromosome harboring the MeCP2 mutation may result in a milder or asymptomatic form of RTT. We have discovered allelic choice trans-factors (Ctcf and Yy1) for XCI. Our intent is to study how these factors regulate the wild type Mecp2 and we are attempting to re-activate the wild type Mecp2 gene using a RTT mouse model. Our studies provide a paradigm for allelic diseases such as X-linked mental retardation, autism, Prader Willi, and Angelman syndromes.
National Institutes of Health
5R01MH090267 — Allelic Choice in Rett Syndrome
Thomas and Agnes Carvel Foundation
Yasunao Kamikawa, Ph.D.
Hugo Pinto, Ph.D.
Tao Wu, Ph.D.