The role of DNA sequence and nucleosome positioning in higher-order chromatin folding

Chromatin is the substrate for all DNA-associated processes and revealing its structure is key to understanding the regulation of nuclear functions. DNA sequence influences the primary positioning and binding affinity of histone octamers, but how this affects higher-order chromatin folding and dynamics is not well understood. Many in vitro chromatin structure studies utilise the Widom 601 DNA template, which strongly positions nucleosomes, and when reconstituted in the presence of linker histones folds into regular fibres approximately 30 nm in diameter. However, as this template uses a synthetic tandem repeat with strong nucleosome positioning properties that are not commonly found in vivo it does not sample the sequence complexity within cells. To explore the properties of more physiological DNA sequences, analogous to what may be observed in cells, I utilised novel DNA templates that contain 25 unique nucleosome positioning sequences derived from the ovine β- lactoglobulin (BLG) gene. Using MNase-seq, BLG sequences were found to position nucleosomes weakly and form irregularly spaced nucleosome arrays. Structural analysis using sucrose gradient sedimentation and small angle X- ray scattering showed that non-repetitive fibres formed disrupted and heterogeneous structures when folded in the presence of the H5 linker histone. Electron microscopy analysis of fibres lacking the H3/H4 tails indicated that non-repetitive arrays have a different folding path compared to 601 fibres. Next, the mechanical properties of the fibres were examined using single- molecule force spectroscopy with magnetic tweezers. This suggested that 601 and non-601 fibres had similar unfolding dynamics in the absence of the H3/H4 tails, and that fibres were fragile under tension. Together, my findings indicate that DNA sequence heterogeneity contributes to chromatin structure variability observed in vivo and can reconcile some of the divergent data observed between in vitro and in vivo studies

Identification of novel genomic imbalances in Saudi patients with congenital heart disease

Abstract Background Quick genetic diagnosis of a patient with congenital heart disease (CHD) is quite important for proper health care and management. Copy number variations (CNV), chromosomal imbalances and rearrangements have been frequently associated with CHD. Previously, due to limitations of microscope based standard karyotyping techniques copious CNVs and submicroscopic imbalances could not be detected in numerous CHD patients. The aim of our study is to identify cytogenetic abnormalities among the selected CHD cases (n = 17) of the cohort using high density oligo arrays. Results Our screening study indicated that six patients (~35%) have various cytogenetic abnormalities. Among the patients, only patient 2 had a duplication whereas the rest carried various deletions. The patients 1, 4 and 6 have only single large deletions throughout their genome; a 3.2 Mb deletion on chromosome 7, a 3.35 Mb deletion on chromosome 3, and a 2.78 Mb a deletion on chromosome 2, respectively. Patients 3 and 5 have two deletions on different chromosomes. Patient 3 has deletions on chromosome 2 (2q24.1; 249 kb) and 16 (16q22.2; 1.8 Mb). Patient 4 has a 3.35 Mb an interstitial deletion on chromosome 3 (3q13.2q13.31). Based on our search on the latest available literature, our study is the first inclusive array CGH evaluation on Saudi cohort of CHD patients. Conclusions This study emphasizes the importance of the arrays in genetic diagnosis of CHD. Based on our results the high resolution arrays should be utilized as first-tier diagnostic tool in clinical care as suggested before by others. Moreover, previously evaluated negative CHD cases (based on standard karyotyping methods) should be re-examined by microarray based cytogenetic methods

cGAS-mediated induction of type I interferon due to inborn errors of histone pre-mRNA processing

International audienceInappropriate stimulation or defective negative regulation of the type I interferon response can lead to autoinflammation. In genetically uncharacterized cases of the type I interferonopathy Aicardi-Goutières syndrome, we identified biallelic mutations in LSM11 and RNU7-1, which encode components of the replication-dependent histone pre-mRNA-processing complex. Mutations were associated with the misprocessing of canonical histone transcripts and a disturbance of linker histone stoichiometry. Additionally, we observed an altered distribution of nuclear cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) and enhanced interferon signaling mediated by the cGAS-stimulator of interferon genes (STING) pathway in patient-derived fibroblasts. Finally, we established that chromatin without linker histone stimulates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) production in vitro more efficiently. We conclude that nuclear histones, as key constituents of chromatin, are essential in suppressing the immunogenicity of self-DNA