Kaul, H. | bioRxiv Science

bioRxiv2021

Discrete-to-analog signal conversion in human pluripotent stem cells

During development, state transitions are coordinated through changes in the identity of molecular regulators in a cell state- and dose specific manner. The ability to rationally engineer such functions in human pluripotent stem cells (hPSC) will enable numerous applications in regenerative medicine. Herein we report the generation of synthetic gene circuits that can detect a discrete cell state, and upon state detection, produce fine-tuned effector proteins in a programmable manner. Effectively, these gene circuits convert a discrete (digital-like) cell state into an analog signal by merging AND-like logic integration of endogenous miRNAs (classifiers) with a miRNA-mediated output fine-tuning technology (miSFITs). Using an automated miRNA identification and model-guided circuit optimization approach, we were able to produce robust cell state specific and graded output production in undifferentiated hPSC. We further finely controlled the levels of endogenous BMP4 secretion, which allowed us to document the effect of endogenous factor secretion in comparison to exogenous factor addition on early tissue development using the hPSC-derived gastruloid system. Our work provides the first demonstration of a discrete-to-analog signal conversion circuit operating in living hPSC, and a platform for customized cell state-specific control of desired physiological factors, laying the foundation for programming cell compositions in hPSC-derived tissues and beyond. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/467377v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@150da71org.highwire.dtl.DTLVardef@135554borg.highwire.dtl.DTLVardef@72b5b3org.highwire.dtl.DTLVardef@f70f84_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2020

Gene regulatory network (GRN) embedded agents connect cellular decision making to human pluripotent stem cell derived germ layer-like pattern formation

The emergence of germ layers in embryos during gastrulation is a key developmental milestone. How morphogenetic signals engage the regulatory networks responsible for early embryonic tissue patterning is incompletely understood. To understand this, we developed a gene regulatory network (GRN) model of human pluripotent stem cell (hPSC) lineage commitment and embedded it into cellular agents that respond to a dynamic signalling microenvironment. We found that cellular pattern order, composition, and dynamics were predictably manipulable based on the GRN wiring. We showed that feedback between OCT4, and BMP and WNT pathways created a dynamic OCT4 front that mediates the spatiotemporal evolution of developmental patterns. Translocation of this radial front can be predictively disrupted in vitro to control germ-layer pattern composition. This work links the emergence of multicellular patterns to regulatory network activity in individual hPSCs. We anticipate our approach will help to understand how GRN structure regulates organogenesis in different contexts.

developmental biology↗

bioRxiv2019

NAD+-dependent deacetylase SIRT1 is essential for meiotic progression and controls repair-recombination efficiency

Meiotic components and their functions have been extensively studied. Yet, the interplay between molecular factors and regulation of their functions that is brought about by post-translational modifications, specifically (de)-acetylation, is not well characterized. SIRT1, a NAD+-dependent deacetylase has been previously shown to be necessary for spermatogenesis. However, whether it has any role to play in mammalian meiosis remains to be uncovered. Our findings identify SIRT1 as a key determinant of meiotic progression. Knocking out SIRT1 specifically in meiocytes (SIRT1{Delta}meio) led to a delay in progression through pachytene and repair of double strand breaks. Interestingly, despite these deficits, meiotic loss of SIRT1 did not affect synapsis nor did it lead to pachytene arrest or apoptosis. Moreover, our results demonstrate that SIRT1 is required for regulating crossover frequency and its absence results in higher crossover events. Therefore, our study brings to the fore a novel regulatory factor/mechanism that is necessary for coupling of synapsis and recombination. This is noteworthy since mutations in core meiotic components result in gross defects in synapsis, repair and recombination, and very few studies have reported the differential regulation of these processes. Further, exposing SIRT1{Delta}meio to low/moderate doses of (C)-irradiation indicated that SIRT1 might be involved in eliciting recombination checkpoint arrest and in its absence pachytene cells progress to diplotene stage, unlike in the SIRT1WT mice. Importantly, exogenous damage resulted in enhanced retention of (C)H2AX in SIRT1{Delta}meio diplotene cells, reiterating the critical role that SIRT1 plays in regulating repair efficiency/kinetics. Molecularly, we find that SIRT1 interacts with MRN complex and lack of SIRT1 causes hyperacetylation of several non-histone proteins including the MRN components. Given that SIRT1{Delta}meio mice mimic MRN hypomorphs, we propose that SIRT1-dependent deacetylation of these proteins is crucial for normal meiotic progression. Taken together, our study uncovers a previously unappreciated role of SIRT1 in meiotic progression.\n\nAuthor SummaryMeiosis is a key process in germ cell development that is essential for generating genetic diversity via recombination. It involves precise spatio-temporal orchestration of various molecular events such as chromosomal synapsis, repair and recombination. Whereas the core meiotic components are well known, upstream factors that might be important for regulating their functions and also couple the downstream processes are less explored. In this paper, we report that SIRT1, a NAD+-dependent deacetylase, is necessary for meiotic progression by identifying its role in coupling of synapsis and recombination. By generating a meiosis specific knockout of SIRT1, we show that its absence in spermatocytes leads to inefficient/delayed repair and progression through pachytene. We have also uncovered that SIRT1 exerts control over recombination (cross over) frequency. Interestingly, our findings demonstrate that SIRT1 provides protection against exogenous genotoxic stress possibly by eliciting meiotic checkpoints. Thus, this study provides both cellular and molecular insights into the importance of SIRT1 mediated protein deacetylation in governing meiosis in mammals.

developmental biology↗