Mutations in the LMNA gene, encoding lamin A/C (LMNA) protein, cause a diverse array of diseases
referred to as laminopathies. Dilated cardiomyopathy (DCM) is common and the main cause of death in
laminopathies. LMNA mutations are also the second most common causes of familial DCM. DCM due to LMNA
mutations (hereafter, LMNA-DCM) has a poor prognosis and a high incidence of sudden cardiac death. The
underpinning mechanism(s) of LMNA-DCM is unknown. Hence, there is no effective therapy for LMNA-DCM.
We have shown that LMNA binds to about 300 genomic regions in human cardiac myocytes, which are
referred to as Lamin-Associated Domains (LADs). LADs comprise about 20% of the genome and several
hundred genes. We show that LADs are redistributed in LMNA-DCM, resulting in Gain of LADs (GoL) and Loss
of LADs (LoL). LoL is associated with active transcription, whereas GoL suppresses gene expression.
At the mechanistic level, Preliminary data show that LADs are lost at the binding motifs for CTCF protein.
CTCF insulates transcriptionally active chromatin loops and recruits topoisomerase 2B (TOP2B) to cut the
compact chromatin and open the loops for active transcription. In parallel with these findings, we show that
double stranded DNA breaks (DSBs), induced by TOP2B, are increased. We also show that expression of DSB
repair genes is suppressed in LMNA-DCM, partly because of GoL. Consequently, DSBs are released into the
cytoplasm and sensed by CGAS protein, which activates DNA damage response (DDR) and expression of genes
involved in cell death, senescence, fibrosis, and cardiac dysfunction, phenotypic features of human LMNA-DCM.
We propose to study the mechanisms responsible for increased DSBs and impaired DSB repair, and
determine therapeutic effects of targeting the DDR pathway in LMNA-DCM. In aim 1, we will test the hypothesis
that LoL leads to active transcription, induction of DSBs, and stalled TOP2B, whereas GoL suppresses
transcription (hence, no DSBs). To test this hypothesis, we will map genome-wide DSB sites at the nucleotide
level by END-Seq technique, compare distribution density of DSBs at LoL, GoL, and non-LAD regions in control
and LMNA-DCM hearts, and determine stalling of TOP2B at the DSBs by ChIP-Seq. In aim 2, we will test the
hypothesis that DSB repair is impaired in LMNA-DCM partly because GoL suppresses expression of key repair
genes and in. part because recruitment of the repair enzymes to the DSB sites is impaired. Recruitment and
assembly of the selected repair proteins at the DSBs will be analyzed by ChIP-Seq to map the binding motifs to
DSB sites. Interactions between LMNA and the repair proteins will be determined by immunoprecipitation. In aim
3, the DDR pathway will be blocked genetically and pharmacologically by inhibiting CGAS, the key sensor of the
cytoplasmic DNA, in two mouse models of LMNA-DCM. The ensuing effects on survival, cardiac function, gene
expression, DDR activation, fibrosis, senescence, and apoptosis will be determined. The findings could delineate
the mechanisms of increased DSBs and determine therapeutic effects of targeting of the DDR in LMNA-DCM.