Ahmet M. Denli

Differential L1 regulation in pluripotent stem cells of humans and apes

Identifying cellular and molecular differences between human and non-human primates (NHPs) is essential to the basic understanding of the evolution and diversity of our own species. Until now, preserved tissues have been the main source for most comparative studies between humans, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus). However, these tissue samples do not fairly represent the distinctive traits of live cell behaviour and are not amenable to genetic manipulation. We propose that induced pluripotent stem (iPS) cells could be a unique biological resource to determine relevant phenotypical differences between human and NHPs, and that those differences could have potential adaptation and speciation value. Here we describe the generation and initial characterization of iPS cells from chimpanzees and bonobos as new tools to explore factors that may have contributed to great ape evolution. Comparative gene expression analysis of human and NHP iPS cells revealed differences in the regulation of long interspersed element-1 (L1, also known as LINE-1) transposons. A force of change in mammalian evolution, L1 elements are retrotransposons that have remained active during primate evolution. Decreased levels of L1-restricting factors APOBEC3B (also known as A3B) and PIWIL2 (ref. 7) in NHP iPS cells correlated with increased L1 mobility and endogenous L1 messenger RNA levels. Moreover, results from the manipulation of A3B and PIWIL2 levels in iPS cells supported a causal inverse relationship between levels of these proteins and L1 retrotransposition. Finally, we found increased copy numbers of species-specific L1 elements in the genome of chimpanzees compared to humans, supporting the idea that increased L1 mobility in NHPs is not limited to iPS cells in culture and may have also occurred in the germ line or embryonic cells developmentally upstream to germline specification during primate evolution. We propose that differences in L1 mobility may have differentially shaped the genomes of humans and NHPs and could have continuing adaptive significance.

Conversion of human fibroblasts to angioblast-like progenitor cells

Lineage conversion of one somatic cell type to another is an attractive approach for generating specific human cell types. Lineage conversion can be direct, in the absence of proliferation and multipotent progenitor generation, or indirect, by the generation of expandable multipotent progenitor states. We report the development of a reprogramming methodology in which cells transition through a plastic intermediate state, induced by brief exposure to reprogramming factors, followed by differentiation. We use this approach to convert human fibroblasts to mesodermal progenitor cells, including by non-integrative approaches. These progenitor cells demonstrated bipotent differentiation potential and could generate endothelial and smooth muscle lineages. Differentiated endothelial cells exhibited neo-angiogenesis and anastomosis in vivo. This methodology for indirect lineage conversion to angioblast-like cells adds to the armamentarium of reprogramming approaches aimed at the study and treatment of ischemic pathologies.

SRY-box-containing Gene 2 Regulation of Nuclear Receptor Tailless (Tlx) Transcription in Adult Neural Stem Cells

Background: Sox2 and TLX are essential for the self-renewal of adult neural stem cells (NSCs). Results: Sox2 positively regulates TLX expression and antagonizes the negative feedback system of TLX by a physical interaction between Sox2 and TLX. Conclusion: Sox2 and TLX form a molecular network regulating adult NSCs. Significance: This molecular network is a target to discover new ways regulate endogenous neurogenesis. Adult neurogenesis is maintained by self-renewable neural stem cells (NSCs). Their activity is regulated by multiple signaling pathways and key transcription factors. However, it has been unclear whether these factors interplay with each other at the molecular level. Here we show that SRY-box-containing gene 2 (Sox2) and nuclear receptor tailless (TLX) form a molecular network in adult NSCs. We observed that both Sox2 and TLX proteins bind to the upstream region of Tlx gene. Sox2 positively regulates Tlx expression, whereas the binding of TLX to its own promoter suppresses its transcriptional activity in luciferase reporter assays. Such TLX-mediated suppression can be antagonized by overexpressing wild-type Sox2 but not a mutant lacking the transcriptional activation domain. Furthermore, through regions involved in DNA-binding activity, Sox2 and TLX physically interact to form a complex on DNAs that contain a consensus binding site for TLX. Finally, depletion of Sox2 revealed the potential negative feedback loop of TLX expression that is antagonized by Sox2 in adult NSCs. These data suggest that Sox2 plays an important role in Tlx transcription in cultured adult NSCs.

The enemy within: intronic miR-26b represses its host gene, ctdsp2, to regulate neurogenesis

Differentiation of multipotent stem cells occurs through the highly coordinated control of gene expression. Repressor element 1 (RE1) silencing transcription factor (REST), a master transcriptional regulator in neuronal stem cells, restricts neuronal gene expression. REST activity is context-dependent and is modified by its cofactors, such as Ctdsp2. In this issue of Genes & Development, Dill and colleagues (pp. 25-30) report on the microRNA-mediated regulation of neural differentiation. Interestingly, this microRNA is post-transcriptionally regulated and modulates expression of its host gene, ctdsp2.

miR-9 and TLX: chasing tails in neural stem cells

Development and maintenance of an organism require the precise spatiotemporal orchestration of stem cell proliferation and differentiation. In neurogenesis, a microRNA and an orphan nuclear receptor comprise a negative feedback loop that regulates neural stem cell fate.

Differentiation of Inflammation-Responsive Astrocytes from Glial Progenitors Generated from Human Induced Pluripotent Stem Cells

Summary Astrocyte dysfunction and neuroinflammation are detrimental features in multiple pathologies of the CNS. Therefore, the development of methods that produce functional human astrocytes represents an advance in the study of neurological diseases. Here we report an efficient method for inflammation-responsive astrocyte generation from induced pluripotent stem cells (iPSCs) and embryonic stem cells. This protocol uses an intermediate glial progenitor stage and generates functional astrocytes that show levels of glutamate uptake and calcium activation comparable with those observed in human primary astrocytes. Stimulation of stem cell-derived astrocytes with interleukin-1β or tumor necrosis factor α elicits a strong and rapid pro-inflammatory response. RNA-sequencing transcriptome profiling confirmed that similar gene expression changes occurred in iPSC-derived and primary astrocytes upon stimulation with interleukin-1β. This protocol represents an important tool for modeling in-a-dish neurological diseases with an inflammatory component, allowing for the investigation of the role of diseased astrocytes in neuronal degeneration.

REST Regulates Non-Cell-Autonomous Neuronal Differentiation and Maturation of Neural Progenitor Cells via Secretogranin II.

RE-1 silencing transcription factor (REST), a master negative regulator of neuronal differentiation, controls neurogenesis by preventing the differentiation of neural stem cells. Here we focused on the role of REST in the early steps of differentiation and maturation of adult hippocampal progenitors (AHPs). REST knockdown promoted differentiation and affected the maturation of rat AHPs. Surprisingly, REST knockdown cells enhanced the differentiation of neighboring wild-type AHPs, suggesting that REST may play a non–cell-autonomous role. Gene expression analysis identified Secretogranin II (Scg2) as the major secreted REST target responsible for the non–cell-autonomous phenotype. Loss-of-function of Scg2 inhibited differentiation in vitro, and exogenous SCG2 partially rescued this phenotype. Knockdown of REST in neural progenitors in mice led to precocious maturation into neurons at the expense of mushroom spines in vivo. In summary, we found that, in addition to its cell-autonomous function, REST regulates differentiation and maturation of AHPs non–cell-autonomously via SCG2. SIGNIFICANCE STATEMENT Our results reveal that REST regulates differentiation and maturation of neural progenitor cells in vitro by orchestrating both cell-intrinsic and non–cell-autonomous factors and that Scg2 is a major secretory target of REST with a differentiation-enhancing activity in a paracrine manner. In vivo, REST depletion causes accelerated differentiation of newborn neurons at the expense of spine defects, suggesting a potential role for REST in the timing of the maturation of granule neurons.

A Novel Model for an Older Remodeler: The BAF Swap in Neurogenesis

Changes in chromatin state contribute to the switch in gene expression programs that characterizes the transition of dividing neural stem cells toward a neuronal fate. In this issue of Neuron, Lessard et al. show that this process is regulated by specific cofactor exchanges within the SWI/SNF chromatin remodeling complex.

High-resolution comparative analysis of great ape genomes

A spotlight on great ape genomes Most nonhuman primate genomes generated to date have been “humanized” owing to their many gaps and the reliance on guidance by the reference human genome. To remove this humanizing effect, Kronenberg et al. generated and assembled long-read genomes of a chimpanzee, an orangutan, and two humans and compared them with a previously generated gorilla genome. This analysis recognized genomic structural variation specific to humans and particular ape lineages. Comparisons between human and chimpanzee cerebral organoids showed down-regulation of the expression of specific genes in humans, relative to chimpanzees, related to noncoding variation identified in this analysis. Science, this issue p. eaar6343 Analysis of long-read great ape and human genomes identifies human-specific changes affecting brain gene expression. INTRODUCTION Understanding the genetic differences that make us human is a long-standing endeavor that requires the comprehensive discovery and comparison of all forms of genetic variation within great ape lineages. RATIONALE The varied quality and completeness of ape genomes have limited comparative genetic analyses. To eliminate this contiguity and quality disparity, we generated human and nonhuman ape genome assemblies without the guidance of the human reference genome. These new genome assemblies enable both coarse and fine-scale comparative genomic studies. RESULTS We sequenced and assembled two human, one chimpanzee, and one orangutan genome using high-coverage (>65x) single-molecule, real-time (SMRT) long-read sequencing technology. We also sequenced more than 500,000 full-length complementary DNA samples from induced pluripotent stem cells to construct de novo gene models, increasing our knowledge of transcript diversity in each ape lineage. The new nonhuman ape genome assemblies improve gene annotation and genomic contiguity (by 30- to 500-fold), resulting in the identification of larger synteny blocks (by 22- to 74-fold) when compared to earlier assemblies. Including the latest gorilla genome, we now estimate that 83% of the ape genomes can be compared in a multiple sequence alignment. We observe a modest increase in single-nucleotide variant divergence compared to previous genome analyses and estimate that 36% of human autosomal DNA is subject to incomplete lineage sorting. We fully resolve most common repeat differences, including full-length retrotransposons such as the African ape-specific endogenous retroviral element PtERV1. We show that the spread of this element independently in the gorilla and chimpanzee lineage likely resulted from a founder element that failed to segregate to the human lineage because of incomplete lineage sorting. The improved sequence contiguity allowed a more systematic discovery of structural variation (>50 base pairs in length) (see the figure). We detected 614,186 ape deletions, insertions, and inversions, assigning each to specific ape lineages. Unbiased genome scaffolding (optical maps, bacterial artificial chromosome sequencing, and fluorescence in situ hybridization) led to the discovery of large, unknown complex inversions in gene-rich regions. Of the 17,789 fixed human-specific insertions and deletions, we focus on those of potential functional effect. We identify 90 that are predicted to disrupt genes and an additional 643 that likely affect regulatory regions, more than doubling the number of human-specific deletions that remove regulatory sequence in the human lineage. We investigate the association of structural variation with changes in human-chimpanzee brain gene expression using cerebral organoids as a proxy for expression differences. Genes associated with fixed structural variants (SVs) show a pattern of down-regulation in human radial glial neural progenitors, whereas human-specific duplications are associated with up-regulated genes in human radial glial and excitatory neurons (see the figure). CONCLUSION The improved ape genome assemblies provide the most comprehensive view to date of intermediate-size structural variation and highlight several dozen genes associated with structural variation and brain-expression differences between humans and chimpanzees. These new references will provide a stepping stone for the completion of great ape genomes at a quality commensurate with the human reference genome and, ultimately, an understanding of the genetic differences that make us human. SMRT assemblies and SV analyses. (Top) Contiguity of the de novo assemblies. (Bottom, left to right) For each ape, SVdetection was done against the human reference genome as represented by a dot plot of an inversion). Human-specific SVs, identified by comparing ape SVs and population genotyping (0/0, homozygous reference),were compared to single-cell gene expression differences [range: low (dark blue) to high (dark red)] in primary and organoid tissues. Each heatmap row is a gene that intersects an insertion or deletion (green), duplication (cyan), or inversion (light green). Genetic studies of human evolution require high-quality contiguous ape genome assemblies that are not guided by the human reference. We coupled long-read sequence assembly and full-length complementary DNA sequencing with a multiplatform scaffolding approach to produce ab initio chimpanzee and orangutan genome assemblies. By comparing these with two long-read de novo human genome assemblies and a gorilla genome assembly, we characterized lineage-specific and shared great ape genetic variation ranging from single– to mega–base pair–sized variants. We identified ~17,000 fixed human-specific structural variants identifying genic and putative regulatory changes that have emerged in humans since divergence from nonhuman apes. Interestingly, these variants are enriched near genes that are down-regulated in human compared to chimpanzee cerebral organoids, particularly in cells analogous to radial glial neural progenitors.

Examining non-LTR retrotransposons in the context of the evolving primate brain

Researchers have long sought to understand the genetic basis of the cognitive differences between primates, with particular focus on the human brain. Although all mutational types have worked in concert with evolutionary forces to generate the current human brain, in this review we will explore the impact of mobile elements, specifically non-LTR retrotransposons. Non-LTR retrotransposons have contributed coding and regulatory sequences to the genome throughout evolution. During primate evolution there have been multiple waves of LINE retrotransposition as well as the birth of new mobile elements such as the SINEs Alu and SVA and we will explore what kinds of impacts these may have had on the evolving human brain.