It is well established that at interphase the inactive X chromosome (Xi) adopts a specific 3-dimensional conformation that differs from that of the active X chromosome (Xa). Yet we know very little about how this arrangement is achieved, nor how critical this organization is to maintaining the epigenomic changes that are characteristic of Xi. Human Xi is arranged into at least two distinct types of facultative heterochromatin that occupy approximately a dozen alternating multi-megabase (Mb) domains along the length of the chromosome, that by immunofluorescence gives a striped appearance to the chromosome at metaphase. At interphase heterochromatin of the same type coalesce resulting in Xi arranging itself into two compartments resulting in a bi-partite appearance. Several large (40-400 kilobase) tandem repeat (TR) DNA reside at the boundary between some of these heterochromatin bands on the X chromosome. These TRs adopt an Xi-specific euchromatin organization that is bound by the architectural protein CCCTC-binding factor (CTCF). Despite residing 15-60 Mb apart, the TRs make frequent Xi-specific intra-chromosomal contact. Given this behavior, the TR elements may function as epigenetically regulated Xi-specific chromosome folding elements. Using cutting edge genome engineering tools, the largest of these TRs (DXZ4), has been removed from Xi as the most direct way to test this hypothesis. On the Xa and male X chromosome, DXZ4 is packaged into constitutive heterochromatin, an arrangement that is common to other autosomal TR elements. However, in some male carcinoma cells, DXZ4 has been found to ?flip? its chromatin state to one that resembles that seen on Xi. Furthermore, a similar phenomena has been described for several autosomal TR elements in transformed cells as well as at one autosomal TR that is responsible for a form of muscular dystrophy when in this configuration. The ability of large TR DNA to transition between two distinct configurations, and the fact that one form can mediate new long-range chromatin contacts makes understanding how chromatin states are epigenetically regulated at these sequences important. Experiments described in this proposal seek to address, (1) how chromatin states at the TRs are regulated, with an emphasis on the potential role of associated long noncoding RNAs (lncRNAs) that alter in expression as chromatin states change, (2) assess the impact of DXZ4 loss on the maintenance and organization of Xi, and (3) determine if the introduction of TR elements at different locations on the X is sufficient to establish new long-range contacts. It is anticipated that these studies will assess the function of TR DNA on the X chromosome, as well as provide mechanistic insight into the organization and maintenance of Xi territory and the role of lncRNAs in regulating chromatin at large TR DNA in complex genomes.
It is widely recognized that spatial organization of the genome is intimately linked to gene expression, and that failure to maintain gene silencing is a major contributor to disease and cancer. The inactive X chromosome (Xi) provides an excellent model in which to probe the functional relationship between higher-order organization of chromosome territories and the underlying epigenomic state. Here we seek to explore the role of large tandem repeat (TR) loci on the X, that appear to act as epigenetically regulated structural elements, facilitating the organization and maintenance of Xi. Findings from this study will further our understanding of the maintenance of X chromosome inactivation (an essential female process) and will have direct relevance to our understanding of chromatin plasticity at other disease associated TR loci.
| Westervelt, Natalia; Chadwick, Brian P (2018) Characterization of the ICCE Repeat in Mammals Reveals an Evolutionary Relationship with the DXZ4 Macrosatellite through Conserved CTCF Binding Motifs. Genome Biol Evol 10:2190-2204 |
| Sun, Zhuo; Chadwick, Brian P (2018) Loss of SETDB1 decompacts the inactive X chromosome in part through reactivation of an enhancer in the IL1RAPL1 gene. Epigenetics Chromatin 11:45 |
