 Hi, my name is Wei Guazen. My name is Jianjun Chen. My name is Jianjun Newkirk. We are all from Yokomori Lab, Department of Biochemistry, University of California, Irvine, and today we are going to talk about our work on FSHD. Facial scapular humor muscular dystrophy. For short, FSHD is the third most common inherited muscular dystrophy in the US. The main symptom of FSHD is the progressive atrophy of facial, shoulder, and upper arm muscles. The majority of FSHD is linked to the deletion of the repeat sequence, D4Z4. D4Z4 locates in the subterrometric region of chromosome 4 long arm. Normally, each D4Z4 allele contains 11 to 150 repeats, while for 95% FSHD patients, 1 D4Z4 allele has less than 10 repeats. This is named FSHD1. In a small percentage of F-patients, the D4Z4 repeat number is still normal, but they show similar symptoms. This is so-called FSHD2. Recently, it was found that more than 80% of the FSHD2 patients have mutations in SMCHD1 gene. Previously, our lab showed that D4Z4 contains trimethylation of lysine 9 residue of histone H3, a marker for repressive heterochromatin. Why in both FSHD1 and FSHD2 D4Z4, this modification is significantly decreased, indicating the disruption of D4Z4 heterochromatin in FSHD. With the abolishment of histone H3 lysine 9 trimethylation, the binding of two protein factors, HP1-gamma and cohesin on D4Z4 is also impaired in FSHD. Although the loss of histone H3 lysine 9 trimethylation heterochromatin appears to signify that FSHD is functional significance was unclear. Each D4Z4 repeat harbors a DOX4 open reading frame which encodes for a transcriptional activator protein. DOX4 is upregulated in FSHD muscle cells and overexpression of DOX4 caused muscle differentiation defects. Therefore, DOX4 is considered to play a critical role in allowing FSHD pathogenesis. In order to investigate whether the chromatin state affects DOX4 expression, we use two different methods to perturb the heterochromatin at D4Z4 in human and motorized myoblasts. One is by chemical inhibition of H3K9 trimethylation using ketosing. The other is SHRNA depletion of the methyl transferase, SUVAR39H1. Both methods resulted in decreased H3K9 trimethylation at D4Z4 and also caused an upregulation of DOX4 gene expression. These results indicate that H3K9 trimethylation contributes to repression of DOX4 transcription. Previous studies showed that SMCHD1 binds at the D4Z4 repeat and depletion of SMCHD1 resulted in upregulation of DOX4. Therefore, we tested whether H3K9 trimethylation affects SMCHD1 binding at D4Z4. Again, by both chemical inhibition and SHRNA depletion, we found that decreased H3K9 trimethylation indeed resulted in less SMCHD1 binding at D4Z4. These results suggest that the loss of H3K9 trimethylation at D4Z4 contributes to the loss of SMCHD1 and turns leads to abnormal DOX4 expression in FSHD. Through a sequencing of 280 PCR clones, we found that the PCR products from 4Q and 10Q D4Z4 showed only a two nucleotide difference from one another with no other heterogeneity. In contrast, the D4Z4 homologs and seven other chromosomes were highly heterogeneous with interruptions in the DOX4 open reading frame in 90% of clones. Our results indicate that the D4Z4 repeats on chromosome 4Q and 10Q are unusually well conserved in comparison to other D4Z4 homologs. Using chip assay and QPCR primaries specific to 4Q, 10Q, D4Z4 and other homologs, we found that the loss of histone H3K9 trimethylation and HP1 gamma cohesin binding in FSHD patients is only restricted to 4Q, 10Q, D4Z4 but not on other D4Z4 homologs. This result further indicates the role of 4Q, 10Q, D4Z4 in FSHD pathogenesis. Our study showed for the first time that the D4Z4 heterochromatin plays a role in regulating DOX4 expression. We also showed the genetic and epigenetic distinction between the 4Q, 10Q, D4Z4 and the non-4Q, 10Q homologs which highlights the special role of the 4Q, 10Q, D4Z4 heterochromatin.