Poster Q6 - Compartmentalization and chromatin folding in response to tissue rigidity

Introduction: Stromal stiffness modulates DNA compaction, which introduces changes in chromatin markers – repressive and permissive – to influence the cellular transcriptome. We have been studying the impact of stromal stiffness on mammary epithelial cell (MEC) chromatin organization using synthetic substrates of rigidity that mimic healthy and malignant tissue. Results in Control MEC and Scribble KD MEC, showed that either gene expression, chromatin markers, YAP translocation, and Lamin-associated enriched repressive regions are modified when MECs interact with a stiff stroma.

Materials and

Methods: MCF10A MECs were cultured in DMEM/F12 Ham's mixture supplemented with 5% horse serum, 20 ng/ml EGF, 10 μg/ml insulin, 100 ng/ml cholera toxin, and 0.5 mg/ml hydrocortisone. HEK293T for viral transfection for Scribble KD MCF10A cells were cultured in DMEM supplemented with 10% FBS.

Briefly, Polyacrylamide (PA) gels of different rigidities were made mixing different amount of acrylamide and bis-acrylamide and polymerization was trigger by adding 1% potassium persulfate and TEMED. After one hour of polymerization coverslips were removed and Fibronectin was covalently linked to PA using an NHS-based method.

RNA was isolated using TRIzol followed by chloroform extraction. RNAseq library preparation was performed by the Functional Genomics Laboratory (FGL), a QB3-Berkeley Core Research Facility at UC Berkeley. Libraries were pooled evenly by molarity for sequencing. Raw sequencing data were converted into fastq format, sample-specific files using the Illumina bcl2fastq2 software on the sequencing centers local Linux server system. For CUT&Tag, cells were permeabilized with Dig wash buffer overnight. LAMINB1 antibody was conjugated overnight and subsequently, transposing Tn5 was used to cleave enriched regions.

RNAseq and CUT&Tag fastq files were mapped to the primary assembly of the Gencode v33 human genome using Rsubread (version 2.0.1) and counted using feature- Counts. Lowly expressed genes were filtered out if they did not have at least one count per million (CPM) in at least 4 samples. Data normalization was performed using calcNormFactors in edgeR (ver- sion 3.28.1). Gene ontology was performed using Gage (version 2.36.0) with gene lists from MSigDB version 7.2.

Results, Conclusions, and Discussions: Results

Results showed that tissue rigidity influences gene expression and the organization of chromatin markers (H3K9me and H3K9ac). We then investigated if the changes on gene expression are correlated with the compartmentalization of Lamin-associated enriched repressive regions. By using CUT&Tag, we identified areas across the genome that show different levels of repression. We further genetically manipulated MECs by knocking down the master regulator of polarity and adhesion Scribble. Our preliminary results, indicated that disrupted signaling by the loss of scribble, changes the genetic landscape compared to controls cells, chromatin markers and YAP localization.

In conclusion, gene expression which is accompanied by changes in chromatin organization, is altered by tissue rigidity and time-exposure of cells to mechanical stimui. Our results suggest that Lamin-compartments are associated to accessibility and repression to modify genetic landscape during malignant transformation.

The results presented herein, can help to identify regulatory elements that contribute to malignancy and identify new anti-tumor therapeutic targets.

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