Somatic missense mutations in histone 1 genes occur in ~30% of follicular lymphomas and DLBCL and 85% of Hodgkin's lymphomas, with significant mutual co-occurrence among these alleles, most frequently involving H1C and H1E. We crossed constitutive H1C+/-H1E+/- mice with VavP-Bcl2 transgenic mice and observed significant acceleration of lymphomagenesis (p=0.0001). Lymphoma H1 mutations affect the H1 globular domain or C-terminus. We found that the globular domain mutants fail to insert into chromatin whereas C-ter mutants fail to compact chromatin as shown by atomic force microscopy, in vitro assembled nucleosome arrays, and FRET assays in live cells. Hence both types of mutation confer loss of function.
Constitutive H1C/E knockout mice are healthy and have no overt phenotype. However, immunization with T-cell dependent antigen caused significant GC hyperplasia (p=0.013) and disruption of polarity due to expansion in the number of centrocytes. Notably, H1C/EDKO GC B-cells readily outcompeted WT GC B-cells in mixed chimera experiments indicating that they have superior fitness (p=0.0086). To understand the mechanism through which this occurs we performed RNA-seq in H1C/EDKO GC B-cells which revealed an aberrant gene expression signature composed almost entirely of transcriptional activation (n=721 upregulated and n=61 downregulated q=0.05, LFC=log(1.5)). Strikingly, these same genes are upregulated during induced pluripotency (iPS cell) reprogramming, and are normally silenced during early development by the PRC2 complex (p <0.05 to 1e-05). Indeed histone mass spectrometry showed reduced H3K27me3 (p=0.0003) and increased H3K36me2 (p =0.001) in H1C/EDKO GC B-cells.
This prompted us to extensively characterize the epigenome of purified H1C/EDKO vs WT GC B-cells using High-throughput chromosome conformation capture (Hi-C), ATAC-seq, and ChIP-seq for multiple histone marks. From the topological standpoint the genome is distributed in two compartments: Compartment A, consisting of unpacked chromatin available for gene regulatory processes, and compartment B, which is highly packed, silenced and inaccessible. Beyond this organization, sets of genes are organized into boundary delimited domains called TADs that share regulatory information. Remarkably, the primary effect of H1 loss of function was the shifting of approximately 256 TADs from compartment B to compartment A (there was no A to B shift). These TADs manifested highly significant gain of chromatin accessibility by ATACs-seq and featured reciprocal loss of H3K27me3 and gain of H3K36me2. These "B to A" TADs yielded increased looping connectivity and contained the primitive stem cell genes that were upregulated. Indeed, B to A shifting enabled critical stem cell enhancers to interact with and activate various stem cell genes as shown by v4C (e.g. KLF5, PRDM5, MEIS1, etc). Most remarkably, the 3D architecture of H1C/H1EDKO GC B-cells was similar to that of intermediate stages of iPS cell reprogramming.
Consistent with this finding, H1C/EDKO cells exhibited 4-fold greater reprogramming to the iPS state than WT cells after OKSM transduction, and these iPS cells exhibited reduced H3K27me3, stronger pluripotency potential, and impaired differentiation. This effect was rescued by transduction of WT H1, but not globular or C terminal domain mutant H1. Strikingly, the H1C/EDKO primitive stem cell gene expression signature was highly significantly enriched (NES 1.24, FDR<0.001) in the RNA-seq profiles of independent cohorts of DLBCL patients with histone 1 mutations, as well as H1C+/-H1E+/-;VavP-Bcl2 murine lymphomas. Consistent with acquisition of stem cell characteristics, H1C+/-H1E+/-;VavP-Bcl2 but not VavP-Bcl2 primary lymphoma cells manifested lymphoma initiating functionality after secondary transplantation into recipient animals, consistent with the notion that they may have gained stem cell functionality.
Collectively, we find that H1 isoforms are bona fide lymphoma tumor suppressors. We speculate that H1 mutations are limited to GC derived lymphomas due to the stoichiometric need for H1 proteins in these rapidly dividing cells. To the best of our knowledge these are the first data to implicate disruption of topological packing order of chromatin as a cancer driver mechanism, as well as the first data to provide a mechanism whereby mature B-cells can acquire cancer stem cell-like characteristics.
Disclosures
Melnick: Janssen: Research Funding; Epizyme: Consultancy; Constellation: Consultancy.
The Ink4/Arf locus is a barrier for iPS cell reprogramming