Age-Acquired Downregulation of Lmna Leads to Epigenetic Deregulation and Altered HSPC Function

Hematopoietic stem cells (HSCs) exhibit epigenetic reprogramming and decline in function with aging, and these changes may be predisposing mechanisms for development of clonal hematopoiesis and myeloid malignancies. Our recent study characterizing the epigenetic and transcriptional landscape of young (18-30 years) and aged (65-75 years) human CD34 ⁺CD38 ^- bone marrow (BM) cells identified Lamin-A/C (LMNA) as one of the most downregulated genes with aging (7.9-fold, p=1.9x10 ^-13). LMNA encodes the nuclear lamina protein Lamin-A/C and is mutated in the premature aging disorder Hutchinson-Gilford Progeria Syndrome.

To determine whether downregulation of LMNA contributes to the phenotype of human HSC aging, we knocked down LMNA (LMNA KD) using shRNA in young, mobilized peripheral blood CD34 ⁺ cells. With an average knockdown of 60%, we observed ~33% increase in myeloid colony forming potential (p<0.05). LMNA KD also impaired differentiation in liquid culture as determined by persistence of CD34 expression in twice as many cells as controls (p<0.0001) while induction of myeloid CD11b expression was reduced by ~29% (p<0.05), as well as a trend to reduced erythroid (CD71 ⁺GYPA ⁺) differentiation. To investigate the effects of Lmna loss-of-function in vivo, we conditionally deleted Lmna in mice using Vav-Cre. Loss of Lmna (Lmna KO) had no effect on steady state hematopoiesis in young (10 weeks) or middle-aged (14 months) knockout mice. However, Lmna KO BM cells from middle-aged mice generated 2-fold more total colonies than wild-type, floxed controls (WT=126 vs KO=292, p<0.01).

Given that nuclear architecture provides a layer of epigenetic regulation through chromatin-lamina interactions, we investigated how the epigenome changes in the context of LMNA deficiency. By super-resolution microscopy, we observed loss of LMNA localization from the nuclear periphery in LMNA KD human CD34 ⁺cells (p<0.05). Since lamina-associated domains generally contain regions of repressed chromatin, we stained for histone H3K9me2 and observed that LMNA KD cells displayed increased scattering throughout the nucleus without a change in its density. Next, we performed ATAC-seq and identified 603 open chromatin sites showing changes in accessibility (FDR<0.1). Over 73% of these sites show reduced accessibility and were associated with genes showing reduced expression by RNA-seq. Gene ontology analysis of these sites identified genes associated with heme biosynthetic and cell differentiation processes (FDR<0.005). Moreover, gene set enrichment analysis of the RNA-seq confirmed that downregulated genes were enriched for genes downregulated in our previously reported CD34 ⁺ aging signature (NES=-1.46, FDR=0.001). To determine whether LMNA KD alters the epigenome, we performed ChIP-seq for active (H3K4me3, H3K27ac) and repressive (H3K9me2) histone marks. LMNA KD resulted in significant reduction of 36% of H3K27ac peaks, which mimics our previous observation of marked age-related losses of H3K27ac. Most differential peaks localized to gene promoters (50%) and introns (34%) and include genes involved in nuclear pore organization and gene silencing (FDR<0.001). Notably, for peaks located at intergenic regions, >35% overlapped with active enhancers we reported as lost with aging and were associated with genes involved in regulation of p38 MAPK cascade (enrichment=2.34E ^-5), an important signaling pathway regulating proliferation and differentiation of HSCs and leukemic cells.

Taken together, LMNA deficiency recapitulates features of aging at the functional and epigenetic level. LMNA KD in young, human CD34 ⁺ cells impaired their differentiation while increasing their colony forming potential. Similarly in middle-aged mice, LMNA KO BM cells showed increased colony forming potential. Genomic changes induced by LMNA deficiency include reduced accessibility and gene transcription, accompanied by changes in localization and occupancy of histone H3K9me2 and H3K27ac, respectively. These epigenetic changes affect genes regulating differentiation and signaling pathways. Thus, we have demonstrated that in addition to its structural role, LMNA also contributes to chromatin regulation of hematopoietic pathways important for normal CD34 ⁺ function.