Non-invasive Chromatin Deformation and Measurement of Differential Mechanical Properties in the Nucleus

The nucleus is highly organized to facilitate coordinated gene transcription. Measuring the rheological properties of the nucleus and its sub-compartments will be crucial to understand the principles underlying nuclear organization. Here, we show that strongly localized temperature gradients (approaching 1°C /μm) can lead to substantial intra-nuclear chromatin displacements (>1 μm), while nuclear area and lamina shape remain unaffected. Using particle image velocimetry (PIV), intra-nuclear displacement fields can be calculated and converted into spatio-temporally resolved maps of various strain components. Using this approach, we show that chromatin displacements are highly reversible, indicating that elastic contributions are dominant in maintaining nuclear organization on the time scale of seconds. In genetically inverted nuclei, centrally compacted heterochromatin displays high resistance to deformation, giving a rigid, solid-like appearance. Correlating spatially resolved strain maps with fluorescent reporters in conventional interphase nuclei reveals that various nuclear compartments possess distinct mechanical identities. Surprisingly, both densely and loosely packed chromatin showed high resistance to deformation, compared to medium dense chromatin. Equally, nucleoli display particularly high rigidity and strong local anchoring to heterochromatin. Our results establish how localized temperature gradients can be used to drive nuclear compartments out of mechanical equilibrium to obtain spatial maps of their material responses.

Lactate Suppresses Retroviral Transduction in Cervical Epithelial Cells through DNA-PKcs Modulation

Recently, we have shown the molecular basis for lactate sensing by cervical epithelial cells resulting in enhanced DNA repair processes through DNA-PKcs regulation. Interestingly, DNA-PKcs is indispensable for proper retroviral DNA integration in the cell host genome. According to recent findings, the mucosal epithelium can be efficiently transduced by retroviruses and play a pivotal role in regulating viral release by cervical epithelial cells. This study examined the effects of lactate on lentiviral transduction in cervical cancer cells (HeLa, CaSki, and C33A) and model glioma cell lines (DNA-PKcs proficient and deficient). Our study showed that L- and D-lactate enhanced DNA-PKcs presence in nuclear compartments by between 38 and 63%, which corresponded with decreased lentiviral transduction rates by between 15 and 36%. Changes in DNA-PKcs expression or its inhibition with NU7441 also greatly affected lentiviral transduction efficacy. The stimulation of cells with either HCA1 agonist 3,5-DHBA or HDAC inhibitor sodium butyrate mimicked, in part, the effects of L-lactate. The inhibition of lactate flux by BAY-8002 enhanced DNA-PKcs nuclear localization which translated into diminished lentiviral transduction efficacy. Our study suggests that L- and D-lactate present in the uterine cervix may play a role in the mitigation of viral integration in cervical epithelium and, thus, restrict the viral oncogenic and/or cytopathic potential.

Hi-C-LSTM: Learning representations of chromatin contacts using a recurrent neural network identifies genomic drivers of 3D genome conformation

Despite the availability of chromatin conformation capture experiments, discerning the relationship between the 1D genome and 3D conformation remains a challenge, which limits our understanding of their affect on gene expression and disease. We propose Hi-C-LSTM, a method that produces low-dimensional latent representations that summarize intra-chromosomal Hi-C contacts via a recurrent long short-term memory (LSTM) neural network model. We find that these representations contain all the information needed to recreate the original Hi-C matrix with high accuracy, outperforming existing methods. These representations enable the identification of a variety of conformation-defining genomic elements, including nuclear compartments and conformation-related transcription factors. They furthermore enable in-silico perturbation experiments that measure the influence of cis-regulatory elements on conformation.

Hi-C-LSTM: Learning representations of chromatin contacts using a recurrent neural network identifies genomic drivers of conformation

Despite the availability of chromatin conformation capture experiments, understanding the relationship between regulatory elements and conformation remains a challenge. We propose Hi-C-LSTM, a method that produces low dimensional latent representations that summarize intra-chromosomal Hi-C contacts via a recurrent long short-term memory (LSTM) neural network model. We find that these representations contain all the information needed to recreate the original Hi-C matrix with high accuracy, outperforming existing methods. These representations enable the identification of a variety of conformation-defining genomic elements, including nuclear compartments and conformation-related transcription factors. They furthermore enable in-silico perturbation experiments that measure the influence of cis-regulatory elements on conformation.

The MYH9 Cytoskeletal Protein Is a Novel Corepressor of Androgen Receptors

In the progression of castration-resistant prostate cancer (CRPC), the androgen receptor (AR) that serves as a transcription factor becomes the most remarkable molecule. The transcriptional activity of AR is regulated by various coregulators. As a result, altered expression levels, an aberrant location or activities of coregulators promote the development of prostate cancer. We describe herein results showing that compared with androgen-dependent prostate cancer (ADPC) cells, AR nuclear translocation capability is enhanced in androgen-independent prostate cancer (AIPC) cells. To gain insight into whether AR coregulators are responsible for AR translocation capability, we performed coimmunoprecipitation (CO-IP) coupled with LC-MS/MS to screen 27 previously reported AR cofactors and 46 candidate AR cofactors. Furthermore, one candidate, myosin heavy chain 9 (MYH9), was identified and verified as a novel AR cofactor. Interestingly, the distribution of MYH9 was in both the cytoplasmic and nuclear compartments yet was enriched in the nucleus when AR was knocked down by AR shRNA, suggesting that the nuclear translocation of MYH9 was negatively regulated by AR. In addition, we found that blebbistatin, an inhibitor of MYH9, not only promoted AR nuclear translocation but also enhanced the expression of the AR target gene PSA, which indicates that MYH9 represses nuclear AR signaling. Taken together, our findings reveal that MYH9 appears to be a novel corepressor of AR plays a pivotal role in the progression of CRPC.

Ultrastructural immunocytochemistry shows impairment of RNA pathways in skeletal muscle nuclei of old mice: A link to sarcopenia?

During aging, skeletal muscle is affected by sarcopenia, a progressive decline in muscle mass, strength and endurance that leads to loss of function and disability. Cell nucleus dysfunction is a possible factor contributing to sarcopenia because aging-associated alterations in mRNA and rRNA transcription/maturation machinery have been shown in several cell types including muscle cells. In this study, the distribution and density of key molecular factors involved in RNA pathways namely, nuclear actin (a motor protein and regulator of RNA transcription), 5-methyl cytosine (an epigenetic regulator of gene transcription), and ribonuclease A (an RNA degrading enzyme) were compared in different nuclear compartments of late adult and old mice myonuclei by means of ultrastructural immunocytochemistry. In all nuclear compartments, an age-related decrease of nuclear actin suggested altered chromatin structuring and impaired nucleus-to-cytoplasm transport of both mRNA and ribosomal subunits, while a decrease of 5-methyl cytosine and ribonuclease A in the nucleoli of old mice indicated an age-dependent loss of rRNA genes. These findings provide novel experimental evidence that, in the aging skeletal muscle, nuclear RNA pathways undergo impairment, likely hindering protein synthesis and contributing to the onset and progression of sarcopenia.

Extranuclear Inheritance of Mitochondrial Genome and Epigenetic Reprogrammability of Chromosomal Telomeres in Somatic Cell Cloning of Mammals

The effectiveness of somatic cell nuclear transfer (SCNT) in mammals seems to be still characterized by the disappointingly low rates of cloned embryos, fetuses, and progeny generated. These rates are measured in relation to the numbers of nuclear-transferred oocytes and can vary depending on the technique applied to the reconstruction of enucleated oocytes. The SCNT efficiency is also largely affected by the capability of donor nuclei to be epigenetically reprogrammed in a cytoplasm of reconstructed oocytes. The epigenetic reprogrammability of donor nuclei in SCNT-derived embryos appears to be biased, to a great extent, by the extranuclear (cytoplasmic) inheritance of mitochondrial DNA (mtDNA) fractions originating from donor cells. A high frequency of mtDNA heteroplasmy occurrence can lead to disturbances in the intergenomic crosstalk between mitochondrial and nuclear compartments during the early embryogenesis of SCNT-derived embryos. These disturbances can give rise to incorrect and incomplete epigenetic reprogramming of donor nuclei in mammalian cloned embryos. The dwindling reprogrammability of donor nuclei in the blastomeres of SCNT-derived embryos can also be impacted by impaired epigenetic rearrangements within terminal ends of donor cell-descended chromosomes (i.e., telomeres). Therefore, dysfunctions in epigenetic reprogramming of donor nuclei can contribute to the enhanced attrition of telomeres. This accelerates the processes of epigenomic aging and replicative senescence in the cells forming various tissues and organs of cloned fetuses and progeny. For all the above-mentioned reasons, the current paper aims to overview the state of the art in not only molecular mechanisms underlying intergenomic communication between nuclear and mtDNA molecules in cloned embryos but also intrinsic determinants affecting unfaithful epigenetic reprogrammability of telomeres. The latter is related to their abrasion within somatic cell-inherited chromosomes.

Nuclear upregulation of class I phosphoinositide 3-kinase p110β correlates with high 47S rRNA levels in cancer cells

ABSTRACTThe class I phosphoinositide 3-kinase (PI3K) catalytic subunits p110α and p110β are ubiquitously expressed but differently targeted in tumours. In cancer, PIK3CB (encoding p110β) is seldom mutated compared with PIK3CA (encoding p110α) but can contribute to tumorigenesis in certain PTEN-deficient tumours. The underlying molecular mechanisms are, however, unclear. We have previously reported that p110β is highly expressed in endometrial cancer (EC) cell lines and at the mRNA level in primary patient tumours. Here, we show that p110β protein levels are high in both the cytoplasmic and nuclear compartments in EC cells. Moreover, high nuclear:cytoplasmic staining ratios were detected in high-grade primary tumours. High levels of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] were measured in the nucleus of EC cells, and pharmacological and genetic approaches showed that its production was partly dependent upon p110β activity. Using immunofluorescence staining, p110β and PtdIns(3,4,5)P3 were localised in the nucleolus, which correlated with high levels of 47S pre-rRNA. p110β inhibition led to a decrease in both 47S rRNA levels and cell proliferation. In conclusion, these results present a nucleolar role for p110β that may contribute to tumorigenesis in EC.This article has an associated First Person interview with Fatemeh Mazloumi Gavgani, joint first author of the paper.