Comprehensive genome-wide DNA methylation profiling is critical to gain insights into epigenetic reprogramming during development and disease processes. Among the different genome-wide DNA ...methylation technologies, whole genome bisulphite sequencing (WGBS) is considered the gold standard for assaying genome-wide DNA methylation at single base resolution. However, the high sequencing cost to achieve the optimal depth of coverage limits its application in both basic and clinical research. To achieve 15× coverage of the human methylome, using WGBS, requires approximately three lanes of 100-bp-paired-end Illumina HiSeq 2500 sequencing. It is important, therefore, for advances in sequencing technologies to be developed to enable cost-effective high-coverage sequencing.
In this study, we provide an optimised WGBS methodology, from library preparation to sequencing and data processing, to enable 16-20× genome-wide coverage per single lane of HiSeq X Ten, HCS 3.3.76. To process and analyse the data, we developed a WGBS pipeline (METH10X) that is fast and can call SNPs. We performed WGBS on both high-quality intact DNA and degraded DNA from formalin-fixed paraffin-embedded tissue. First, we compared different library preparation methods on the HiSeq 2500 platform to identify the best method for sequencing on the HiSeq X Ten. Second, we optimised the PhiX and genome spike-ins to achieve higher quality and coverage of WGBS data on the HiSeq X Ten. Third, we performed integrated whole genome sequencing (WGS) and WGBS of the same DNA sample in a single lane of HiSeq X Ten to improve data output. Finally, we compared methylation data from the HiSeq 2500 and HiSeq X Ten and found high concordance (Pearson r > 0.9×).
Together we provide a systematic, efficient and complete approach to perform and analyse WGBS on the HiSeq X Ten. Our protocol allows for large-scale WGBS studies at reasonable processing time and cost on the HiSeq X Ten platform.
Vascular smooth muscle cells (VSMCs) display extraordinary phenotypic plasticity. This allows them to differentiate or dedifferentiate, depending on environmental cues. The ability to ‘switch’ ...between a quiescent contractile phenotype to a highly proliferative synthetic state renders VSMCs as primary mediators of vascular repair and remodelling. When their plasticity is pathological, it can lead to cardiovascular diseases such as atherosclerosis and restenosis. Coinciding with significant technological and conceptual innovations in RNA biology, there has been a growing focus on the role of alternative splicing in VSMC gene expression regulation. Herein, we review how alternative splicing and its regulatory factors are involved in generating protein diversity and altering gene expression levels in VSMC plasticity. Moreover, we explore how recent advancements in the development of splicing-modulating therapies may be applied to VSMC-related pathologies.
Transcription of new RNA is crucial for maintaining synaptic plasticity, learning and memory. Although the importance of synaptic plasticity-related messenger RNAs (mRNAs) is well established, the ...role of a large group of long non-coding RNAs (lncRNAs) in long-term potentiation (LTP) is not known. In this study, we demonstrated the expression of a lncRNA cluster, namely maternally expressed gene 3 (
), retrotransposon-like gene 1-anti-sense (
),
and
, which is located in the maternally imprinted
region on mouse chromosome 12qF1, in primary cortical neurons following glycine stimulation in an N-Methyl-D-aspartate receptor (NMDAR)-dependent manner. Importantly, we also validated the expression of
,
and
in the hippocampus of mice following cued fear conditioning
. Interestingly,
is the only lncRNA that is expressed in the nucleus and cytoplasm. Further analysis revealed that
loss of function blocked the glycine-induced increase of the GluA1 subunit of AMPA receptors on the plasma membrane, a major hallmark of LTP. This aberrant trafficking of AMPA receptors correlated with the dysregulation of the phosphatidylinoside-3-kinase (PI3K)/AKT signaling pathway and the downregulation of the lipid phosphatase and tensin homolog (PTEN). These findings provide the first evidence for a functional role of the lncRNA
in the intricate regulation of the PTEN/PI3K/AKT signaling cascade during synaptic plasticity in neurons.
Conventional dendritic cells (cDCs) are continuously replenished by bone marrow‐derived precursors called pre‐DCs, which traffic through the blood to peripheral tissues. Pre‐DCs are a heterogeneous ...population that includes cDC subset‐committed progenitors, namely pre‐cDC1 and pre‐cDC2, which give rise to mature cDC1 and cDC2, respectively. Regulation of pre‐DC subset trafficking is thought to aid the host response to immune challenge. However, the molecular cues regulating pre‐cDC1 versus pre‐cDC2 trafficking toward peripheral sites during homeostasis and disease remain elusive. Here, we report that pre‐cDC1 but not pre‐cDC2 express the T helper type 1‐associated chemokine receptor CXCR3. Moreover, we identify a cell‐intrinsic role for CXCR3 in the trafficking of pre‐cDC1 to melanoma tumors but not to non‐inflamed organs. We also show that tumor cDC1 numbers can be increased pharmacologically by targeting dipeptidyl peptidase‐4 (CD26), a negative regulator of CXCR3 ligands. Our findings demonstrate that pre‐cDC1 trafficking is regulated distinctly from pre‐cDC2, which is relevant for our understanding of the DC lineage in the context of cancer and inflammation.
The migration of conventional dendritic cell (cDC) precursors (pre‐DCs) is required for anti‐cancer immunity. The two subsets of cDC, cDC1 and cDC2, derive from committed progenitors called pre‐cDC1 and pre‐cDC2, respectively. In this study, we describe the differential expression of chemokine receptors by pre‐DC subsets and show that pre‐cDC1 but not pre‐cDC2 use CXCR3 to traffic into melanoma tumors in mice. We further show that the number of cDC1 within tumors can be increased through pharmacological inhibition of dipeptidyl‐peptidase 4 (DPP4), which normally inactivates the CXCR3 ligands CXCL9 and CXCL10.
Since the discovery of microRNAs (miRNAs) in the early 1990s, these small molecules have been increasingly recognized as key players in the regulation of critical biological processes. They have also ...been implicated in many diverse human diseases. The canonical function of miRNAs is to target the 3' untranslated region (3' UTR) of cytoplasmic messenger RNA to post-transcriptionally regulate mRNA and protein levels. It has now been shown that miRNAs can also bind to the promoter regions of genes or primary miRNA transcripts to regulate gene expression. Such observations have indicated the presence of miRNAs in the nucleus and implied additional non-canonical functions. Nevertheless, the role(s) of nuclear miRNAs in normal hemopoiesis and cancer remains elusive despite a burgeoning literature. Herein, we review current knowledge concerning the abundance and/or functions of nuclear miRNAs during blood cell development and cancer biology. We also discuss ongoing challenges in order to provoke further studies into identifying key roles for nuclear miRNAs in the development of other cell lineages and human cancers.
Activity-dependent gene expression and protein translation underlie the ability of neurons to dynamically adjust their synaptic strength in response to sensory experience and during learning. The ...emerging field of epitranscriptomics (RNA modifications) has rapidly shifted our views on the mechanisms that regulate gene expression. Among hundreds of biochemical modifications on RNA, N6-methyladenosine (m6A) is the most abundant reversible mRNA modification in the brain. Its dynamic nature and ability to regulate all aspects of mRNA processing have positioned m6A as an important and versatile regulator of nervous system functions, including neuronal plasticity, learning and memory. In this review, we summarise recent experimental evidence that supports the role of m6A signalling in learning and memory, as well as providing an overview of the underlying molecular mechanisms in neurons. We also discuss the consequences of perturbed m6A signalling and/or its regulatory networks which are increasingly being linked to various cognitive disorders in humans.
Myelopoiesis involves differentiation of hematopoietic stem cells to cellular populations that are restricted in their self-renewal capacity, beginning with the common myeloid progenitor (CMP) and ...leading to mature cells including monocytes and granulocytes. This complex process is regulated by various extracellular and intracellular signals including microRNAs (miRNAs). We characterised the miRNA profile of human CD34
CD38
myeloid progenitor cells, and mature monocytes and granulocytes isolated from cord blood using TaqMan Low Density Arrays. We identified 19 miRNAs that increased in both cell types relative to the CMP and 27 that decreased. miR-125b and miR-10a were decreased by 10-fold and 100-fold respectively in the mature cells. Using in vitro granulopoietic differentiation of human CD34
cells we show that decreases in both miR-125b and miR-10a correlate with a loss of CD34 expression and gain of CD11b and CD15 expression. Candidate target mRNAs were identified by co-incident predictions between the miRanda algorithm and genes with increased expression during differentiation. Using luciferase assays we confirmed MCL1 and FUT4 as targets of miR-125b and the transcription factor KLF4 as a target of miR-10a. Together, our data identify miRNAs with differential expression during myeloid development and reveal some relevant miRNA-target pairs that may contribute to physiological differentiation.
Mouse kidney parvovirus (MKPV) is a member of the provisional genus Chapparvovirus that causes renal disease in immune-compromised mice, with a disease course reminiscent of polyomavirus-associated ...nephropathy in immune-suppressed kidney transplant patients. Here we map four major MKPV transcripts, created by alternative splicing, to a common initiator region, and use mass spectrometry to identify "p10" and "p15" as novel chapparvovirus accessory proteins produced in MKPV-infected kidneys. p15 and the splicing-dependent putative accessory protein NS2 are conserved in all near-complete amniote chapparvovirus genomes currently available (from mammals, birds and a reptile). In contrast, p10 may be encoded only by viruses with >60% amino acid identity to MKPV. We show that MKPV is kidney-tropic and that the bat chapparvovirus DrPV-1 and a non-human primate chapparvovirus, CKPV, are also found in the kidneys of their hosts. We propose, therefore, that many mammal chapparvoviruses are likely to be nephrotropic.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
