Biology subjects

Henikoff, J. G.

Publications and source records attributed to Henikoff, J. G..

7 recordsLinked to original sources

bioRxiv2025

Superabundant microRNAs are transcribed from human rDNA spacer promoters insulated by CTCF

MicroRNAs are [~]22-nucleotide RNAs processed from primary transcripts and exported from the nucleus to repress gene expression by base-pairing to mRNAs. We find that the highest levels of RNA Polymerase II at human microRNA genes are within the ribosomal gene repeat arrays (rDNAs). Alignment of public nascent transcript data to the hs1 human genome assembly reveals a 50-nucleotide transcript for both miR-1275 and miR-6724, which exits from the nucleus with exceptional rapidity. We show that the miR-1275/miR-6724 transcription unit is closely flanked by CCCTC-binding factor (CTCF) within a <400-bp span of the rDNA spacer promoter. MiR-1275/miR-6724 and microRNA precursors expressed from the 5 External Transcribed Spacer (5ETS) are exported independently of known RNA processing activities and are detected in exosomes and as circulating cancer biomarkers. We propose that rDNA spacer promoter and 5ETS microRNA genes have evolved for general regulatory functions in recipient cells.

genomics↗

bioRxiv2024

Direct measurement of RNA Polymerase II hypertranscription in cancer FFPE samples

Genome-wide hypertranscription is common in hu-man cancer and predicts poor prognosis. To under-stand how hypertranscription might drive cancer, we applied our FFPE-CUTAC method for mapping RNA Polymerase II (RNAPII) genome-wide in formalin-fixed paraffin-embedded (FFPE) sections. We demonstrate global RNAPII elevations in mouse gliomas and assort-ed human tumors in small clinical samples and discov-er regional elevations corresponding to de novo HER2 amplifications punctuated by likely selective sweeps. RNAPII occupancy at replication-coupled histone genes correlated with WHO grade in meningiomas, ac-curately predicted rapid recurrence, and corresponded to whole-arm chromosome losses. Elevated RNAPII at histone genes in meningiomas and diverse breast cancers is consistent with histone production being rate-limiting for S-phase progression and histone gene hypertranscription driving overproliferation and aneu-ploidy in cancer, with general implications for precision oncology.

cancer biology↗

bioRxiv2023

Epigenomic analysis of Formalin-Fixed Paraffin-Embedded samples by CUT&Tag

For more than a century, Formalin Fixed Paraffin Embedded (FFPE) sample preparation has been the preferred method for long-term preservation of biological material. However, the use of FFPE samples for epigenomic studies has been difficult because of chromatin damage from long exposure to high concentrations of formaldehyde. Previously, we introduced Cleavage Under Targeted Accessible Chromatin (CUTAC), an antibody-targeted chromatin accessibility mapping protocol based on CUT&Tag. Here we show that simple modifications of our single-tube CUTAC protocol are sufficient to produce high-resolution maps of paused RNA Polymerase II (RNAPII) at enhancers and promoters using FFPE samples. We find that transcriptional regulatory element differences produced by FFPE-CUTAC distinguish between mouse brain tumor specimens and identify regulatory element markers with high confidence and precision. Our simple work-flow is suitable for automation, making possible affordable epigenomic profiling of archived biological samples for biomarker identification, clinical applications and retrospective studies.

cancer biology↗

bioRxiv2022

A giant virus genome is densely packaged by stable nucleosomes

The two doublet histones of Marseillevirus are distantly related to the four eukaryotic core histones and wrap 121 basepairs of DNA to form remarkably similar nucleosomes. By permeabilizing Marseillevirus virions and performing genome-wide nuclease digestion, chemical cleavage and mass spectrometry assays, we find that the higher-order organization of Marseillevirus chromatin fundamentally differs from that of eukaryotes. Marseillevirus nucleosomes fully protect DNA within virions as closely abutted 121-bp DNA wrapped cores without linker DNA or phasing along genes. Likewise, we observed that a large fraction of the nucleosomes reconstituted onto multi-copy tandem repeats of a nucleosome positioning sequence are tightly packed. Dense promiscuous packing of fully wrapped nucleosomes rather than "beads-on-a-string" with genic punctuation represents a new mode of DNA packaging by histones. We suggest that doublet histones have evolved for viral genome protection and may resemble an early stage of histone differentiation leading to the eukaryotic octameric nucleosome.

evolutionary biology↗

bioRxiv2020

Histone deposition pathways determine the chromatin landscapes of H3.1 and H3.3 K27M oncohistones

Lysine 27-to-methionine (K27M) mutations in the H3.1 or H3.3 histone genes are characteristic of pediatric diffuse midline gliomas (DMGs). These oncohistone mutations dominantly inhibit histone H3K27 trimethylation and silencing, but it is unknown how oncohistone type affects gliomagenesis. We show that the genomic distributions of H3.1 and H3.3 oncohistones in human patient-derived DMG cells are consistent with the DNA replication-coupled deposition of histone H3.1 and the predominant replication-independent deposition of histone H3.3. Although H3K27 trimethylation is reduced for both oncohistone types, H3.3K27M-bearing cells retain some domains, and only H3.1K27M-bearing cells lack H3K27 trimethylation. Neither oncohistone interferes with PRC2 binding. Using Drosophila as a model, we demonstrate that inhibition of H3K27 trimethylation occurs only when H3K27M oncohistones are deposited into chromatin and only when expressed in cycling cells. We propose that oncohistones inhibit the H3K27 methyltransferase as chromatin patterns are being duplicated in proliferating cells, predisposing them to tumorigenesis.

molecular biology↗

bioRxiv2020

Profiling the epigenome at home

Chromatin accessibility mapping is a powerful approach to identify potential regulatory elements. A popular example is ATAC-seq, whereby Tn5 transposase inserts sequencing adapters into accessible DNA ( tagmentation). CUT&Tag is a tagmentation-based epigenomic profiling method in which antibody tethering of Tn5 to a chromatin epitope of interest profiles specific chromatin features in small samples and single cells. Here we show that by simply modifying the tagmentation conditions for histone H3K4me2 or H3K4me3 CUT&Tag, antibody-tethered tagmentation of accessible DNA sites is redirected to produce chromatin accessibility maps that are indistinguishable from the best ATAC-seq maps. Thus, chromatin accessibility maps can be produced in parallel with CUT&Tag maps of other epitopes with all steps from nuclei to amplified sequencing-ready libraries performed in single PCR tubes in the laboratory or on a home workbench. As H3K4 methylation is produced by transcription at promoters and enhancers, our method identifies transcription-coupled accessible regulatory sites.

genomics↗

bioRxiv2019

CUT&Tag for efficient epigenomic profiling of small samples and single cells

Many chromatin features play critical roles in regulating gene expression. A complete understanding of gene regulation will require the mapping of specific chromatin features in small samples of cells at high resolution. Here we describe Cleavage Under Targets and Tagmentation (CUT&Tag), an enzyme-tethering strategy that provides efficient high-resolution sequencing libraries for profiling diverse chromatin components. In CUT&Tag, a chromatin protein is bound in situ by a specific antibody, which then tethers a protein A-Tn5 transposase fusion protein. Activation of the transposase efficiently generates fragment libraries with high resolution and exceptionally low background. All steps from live cells to sequencing-ready libraries can be performed in a single tube on the benchtop or a microwell in a high-throughput pipeline, and the entire procedure can be performed in one day. We demonstrate the utility of CUT&Tag by profiling histone modifications, RNA Polymerase II and transcription factors on low cell numbers and single cells.

genomics↗