Junjie Lu

Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids.

Human-pluripotent-stem-cell-derived kidney cells (hPSC-KCs) have important potential for disease modelling and regeneration. Whether the hPSC-KCs can reconstitute tissue-specific phenotypes is currently unknown. Here we show that hPSC-KCs self-organize into kidney organoids that functionally recapitulate tissue-specific epithelial physiology, including disease phenotypes after genome editing. In three-dimensional cultures, epiblast-stage hPSCs form spheroids surrounding hollow, amniotic-like cavities. GSK3β inhibition differentiates spheroids into segmented, nephron-like kidney organoids containing cell populations with characteristics of proximal tubules, podocytes and endothelium. Tubules accumulate dextran and methotrexate transport cargoes, and express kidney injury molecule-1 after nephrotoxic chemical injury. CRISPR/Cas9 knockout of podocalyxin causes junctional organization defects in podocyte-like cells. Knockout of the polycystic kidney disease genes PKD1 or PKD2 induces cyst formation from kidney tubules. All of these functional phenotypes are distinct from effects in epiblast spheroids, indicating that they are tissue specific. Our findings establish a reproducible, versatile three-dimensional framework for human epithelial disease modelling and regenerative medicine applications.

Replication timing and transcriptional control: beyond cause and effect―part II

Replication timing is frequently discussed superficially in terms of its relationship to transcriptional activity via chromatin structure. However, so little is known about what regulates where and when replication initiates that it has been impossible to identify mechanistic and causal relationships. Moreover, much of our knowledge base has been anecdotal, derived from analyses of a few genes in unrelated cell lines. Recent studies have revisited long-standing hypotheses using genome-wide approaches. In particular, the foundation of this field was recently shored up with incontrovertible evidence that cellular differentiation is accompanied by coordinated changes in replication timing and transcription. These changes accompany subnuclear repositioning, and take place at the level of megabase-sized domains that transcend localized changes in chromatin structure or transcription. Inferring from these results, we propose that there exists a key transition during the middle of S-phase and that changes in replication timing traversing this period are associated with subnuclear repositioning and changes in the activity of certain classes of promoters.

Proliferation-dependent and cell cycle–regulated transcription of mouse pericentric heterochromatin

Pericentric heterochromatin transcription has been implicated in Schizosaccharomyces pombe heterochromatin assembly and maintenance. However, in mammalian systems, evidence for such transcription is inconsistent. We identify two populations of RNA polymerase II–dependent mouse γ satellite repeat sequence–derived transcripts from pericentric heterochromatin that accumulate at different times during the cell cycle. A small RNA species was synthesized exclusively during mitosis and rapidly eliminated during mitotic exit. A more abundant population of large, heterogeneous transcripts was induced late in G1 phase and their synthesis decreased during mid S phase, which is coincident with pericentric heterochromatin replication. In cells that lack the Suv39h1,2 methyltransferases responsible for H3K9 trimethylation, transcription occurs from more sites but is still cell cycle regulated. Transcription is not detected in quiescent cells and induction during G1 phase is sensitive to serum deprivation or the cyclin-dependent kinase inhibitor roscovatine. We demonstrate that mammalian pericentric heterochromatin transcription is linked to cellular proliferation. Our data also provide an explanation for inconsistencies in the detection of such transcripts in different systems.

G9a selectively represses a class of late-replicating genes at the nuclear periphery

We have investigated the role of the histone methyltransferase G9a in the establishment of silent nuclear compartments. Following conditional knockout of the G9a methyltransferase in mouse ESCs, 167 genes were significantly up-regulated, and no genes were strongly down-regulated. A partially overlapping set of 119 genes were up-regulated after differentiation of G9a-depleted cells to neural precursors. Promoters of these G9a-repressed genes were AT rich and H3K9me2 enriched but H3K4me3 depleted and were not highly DNA methylated. Representative genes were found to be close to the nuclear periphery, which was significantly enriched for G9a-dependent H3K9me2. Strikingly, although 73% of total genes were early replicating, more than 71% of G9a-repressed genes were late replicating, and a strong correlation was found between H3K9me2 and late replication. However, G9a loss did not significantly affect subnuclear position or replication timing of any non-pericentric regions of the genome, nor did it affect programmed changes in replication timing that accompany differentiation. We conclude that G9a is a gatekeeper for a specific set of genes localized within the late replicating nuclear periphery.

Space and time in the nucleus: developmental control of replication timing and chromosome architecture.

All eukaryotic cells replicate segments of their genomes in a defined temporal sequence. In multicellular organisms, at least half of the genome is subject to changes in this temporal sequence during development. We now know that this temporal sequence and its developmentally regulated changes are conserved across distantly related species, suggesting that it either represents or reflects something biologically important. However, both the mechanism and the significance of this program remain unknown. We recently demonstrated a remarkably strong genome-wide correlation between replication timing and chromatin interaction maps, stronger than any other chromosomal property analyzed to date, indicating that sequences localized close to one another replicate at similar times. This provides molecular confirmation of long-standing cytogenetic evidence for spatial compartmentalization of early- and late-replicating DNA and supports our earlier model that replication timing is reestablished in each G(1) phase, coincident with the anchorage of chromosomal segments at specific locations within the nucleus (timing decision point [TDP]). Here, we review the evidence linking the replication program to the three-dimensional architecture of chromatin in the nucleus and discuss what such a link might mean for the mechanism and significance of a developmentally regulated replication program.

The inhibition of TDP-43 mitochondrial localization blocks its neuronal toxicity

Genetic mutations in TAR DNA-binding protein 43 (TARDBP, also known as TDP-43) cause amyotrophic lateral sclerosis (ALS), and an increase in the presence of TDP-43 (encoded by TARDBP) in the cytoplasm is a prominent histopathological feature of degenerating neurons in various neurodegenerative diseases. However, the molecular mechanisms by which TDP-43 contributes to ALS pathophysiology remain elusive. Here we have found that TDP-43 accumulates in the mitochondria of neurons in subjects with ALS or frontotemporal dementia (FTD). Disease-associated mutations increase TDP-43 mitochondrial localization. In mitochondria, wild-type (WT) and mutant TDP-43 preferentially bind mitochondria-transcribed messenger RNAs (mRNAs) encoding respiratory complex I subunits ND3 and ND6, impair their expression and specifically cause complex I disassembly. The suppression of TDP-43 mitochondrial localization abolishes WT and mutant TDP-43-induced mitochondrial dysfunction and neuronal loss, and improves phenotypes of transgenic mutant TDP-43 mice. Thus, our studies link TDP-43 toxicity directly to mitochondrial bioenergetics and propose the targeting of TDP-43 mitochondrial localization as a promising therapeutic approach for neurodegeneration.

G2 phase chromatin lacks determinants of replication timing

Chromatin spatial organization helps establish the replication timing decision point at early G1. However, at G2, although retained, chromatin organization is no longer necessary or sufficient to maintain the replication timing program.

Cell cycle regulated transcription of heterochromatin in mammals vs. fission yeast: Functional conservation or coincidence?

Although it is tempting to speculate that the transcription-dependent heterochromatin assembly pathway found in fission yeast may operate in higher mammals, transcription of heterochromatin has been difficult to substantiate in mammalian cells. We recently demonstrated that transcription from the mouse pericentric heterochromatin major (γ) satellite repeats is under cell cycle control being sharply down regulated at the metaphase to anaphase transition and resuming in late G1-phase dependent upon passage through the restriction point. The highest rates of transcription were in early S-phase and again in mitosis with different RNA products detected at each of these times.1 Importantly, differences in the percentage of cells in G1-phase can account for past discrepancies in the detection of major satellite transcripts and suggest that pericentric heterochromatin transcription takes place in all proliferating mammalian cells. A similar cell-cycle regulation of heterochromatin transcription has now been shown in fission yeast,2,3 providing further support for a conserved mechanism. However, there are still fundamental differences between these two systems that preclude the identification of a functional or mechanistic link.

The Distribution of Genomic Variations in Human iPSCs Is Related to Replication-Timing Reorganization during Reprogramming

Cell-fate change involves significant genome reorganization, including changes in replication timing, but how these changes are related to genetic variation has not been examined. To study how a change in replication timing that occurs during reprogramming impacts the copy-number variation (CNV) landscape, we generated genome-wide replication-timing profiles of induced pluripotent stem cells (iPSCs) and their parental fibroblasts. A significant portion of the genome changes replication timing as a result of reprogramming, indicative of overall genome reorganization. We found that early- and late-replicating domains in iPSCs are differentially affected by copy-number gains and losses and that in particular, CNV gains accumulate in regions of the genome that change to earlier replication during the reprogramming process. This differential relationship was present irrespective of reprogramming method. Overall, our findings reveal a functional association between reorganization of replication timing and the CNV landscape that emerges during reprogramming.

Influence of ATM-mediated DNA damage response on genomic variation in human induced pluripotent stem cells

Genome instability is a potential limitation to the research and therapeutic application of induced pluripotent stem cells (iPSCs). Observed genomic variations reflect the combined activities of DNA damage, cellular DNA damage response (DDR), and selection pressure in culture. To understand the contribution of DDR on the distribution of copy number variations (CNVs) in iPSCs, we mapped CNVs of iPSCs with mutations in the central DDR gene ATM onto genome organization landscapes defined by genome-wide replication timing profiles. We show that following reprogramming the early and late replicating genome is differentially affected by CNVs in ATM-deficient iPSCs relative to wild-type iPSCs. Specifically, the early replicating regions had increased CNV losses during retroviral (RV) reprogramming. This differential CNV distribution was not present after later passage or after episomal reprogramming. Comparison of different reprogramming methods in the setting of defective DDR reveals unique vulnerability of early replicating open chromatin to RV vectors.