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Early enforcement of cell identity by a functional component of the terminally differentiated state

03 Jan 2020-bioRxiv (Cold Spring Harbor Laboratory)-

TL;DR: A control principle for robust cell identity is suggested whereby a core component of the differentiated state also promotes differentiation from its own progenitor state, thus providing an explanation for why maintenance of the mature adipocyte state is so robust.
Abstract: How progenitor cells can attain a distinct differentiated cell identity is a challenging problem given that critical transcription factors are often not unique to a differentiation process and the fluctuating signaling environment in which cells exist. Here we test the hypothesis that a unique differentiated cell identity can result from a core component of the differentiated state doubling up as a signaling protein that also drives differentiation. Using live single-cell imaging in the adipocyte differentiation system, we show that progenitor fat cells (preadipocytes) can only commit to terminally differentiate after upregulating FABP4, a lipid buffer that is highly enriched in mature adipocytes. Upon induction of adipogenesis, we show that after a long delay, cells first abruptly start to engage a positive feedback between CEBPA and PPARG before then engaging, after a second delay, a positive feedback between FABP4 and PPARG. These sequential positive feedbacks both need to engage in order to drive PPARG levels past the threshold for irreversible differentiation. In the last step before commitment, PPARG transcriptionally increases FABP4 expression while fatty-acid loaded FABP4 binds to and increases PPARG activity. Together, our study suggests a control principle for robust cell identity whereby a core component of the differentiated state also promotes differentiation from its own progenitor state.
Topics: Cellular differentiation (55%), Progenitor cell (53%), Adipogenesis (53%)

Summary (4 min read)

INTRODUCTION

  • Terminal cell differentiation is fundamental for developing, maintaining, and regenerating tissues in all multicellular organisms and is the process by which specialized cells such as adipocytes (fat cells), osteoblasts, neurons, and muscle cells are generated from progenitor cells [1].
  • Since negative feedback mostly prevents cells which have chosen one path from differentiating into alternative cell types, additional regulatory mechanisms must exist that selectively drive cells onto a unique path and allow cells to robustly assume and maintain a specific differentiated cell identity.
  • These observations motivated their study here that FABP4 could provide such a self-reinforcement mechanism for unique cell identity.
  • It is well-established that the expression of FABP4 is induced by PPARG, the master regulator of adipocyte differentiation [22].
  • Also, it is not clear how FABP4 regulates PPARG and .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

RESULTS

  • FABP4 is needed for adipogenesis in vitro and in vivo Previous studies suggested that FABP4 and PPARG are in a positive feedback relationship [18,20,23,25] (Fig 1A).
  • This analysis showed that cells that go on to differentiate according to their PPARG levels (blue traces in Fig 2C, left) also started to increase their FABP4 levels before they reach the threshold and then increase much more strongly after they reach the threshold (red traces in Fig 2C, right).
  • Previous studies using bulk-cell approaches such as Western blots to quantify FABP4 expression during adipogenesis lacked the sensitivity to observe the small early FABP4 increase that occurs only in the subset of cells that differentiate which is likely the reason why it has been commonly thought that FABP4 is only downstream of PPARG and is induced late in adipogenesis after the critical signaling events for differentiation have already happened [34].
  • Since FABP4 can directly bind to PPARG in the presence of lipid , it is conceivable that the binding may increase PPARG expression indirectly by stabilizing PPARG.
  • In condition 1 (Fig 6D), the authors compared wildtype OP9 preadipocyte cells which expressed FABP4 versus to OP9 cells in which FABP4 had been knocked out (FABP4-KO cells from Fig. 1D).

DISCUSSION

  • The authors study focuses on a general question how cells ensure that they assume and maintain a specific terminally differentiated state given that differentiating cells typically have multiple cell-fate options and there is also cellular plasticity with cells being able to transition between different reversible progenitor states.
  • The authors rationale for focusing on FABP4 was previous evidence that FABP4 may regulate PPARG expression and/or activity [18,20,23,25] and that FABP4 is highly expressed specifically in adipocytes [15].
  • CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
  • The authors study further addressed the question of how FABP4 can regulate PPARG since previous studies have yielded contradictory results.
  • This first appeared to contradict earlier results which showed that WT cells, FABP4/FABP5 KO cells, FABP4/FABP5 KO cells can still effectively differentiate when rosiglitazone was added along with the DMI.

AUTHOR CONTRIBUTIONS

  • Z.B., S.T., A.R., M.L.Z., and M.N.T. designed and carried out experiments and analyzed data.
  • F.B.K. designed experiments and contributed to the discussion.
  • Z.B., T.C., and M.N.T. wrote the manuscript with input from all authors.

DECLARATION OF INTERESTS

  • The authors declare no competing interests.
  • CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

FIGURE LEGENDS

  • (A) Schematic of proposed cell-identity positive feedback.
  • Wildtype 3T3-F442A cells that were transfected and sorted at the same time but that did not harbor FABP4 or FABP5 knockdown were used as a control (Control-KO).
  • (B) Changes in citrine-PPARG and FABP4-mKate expression measured in the same cell over the timecourse of adipogenesis.
  • The timepoint when the feedbacks start to engage is reflected in a change in PPARG slope and occurs many hours before a cell will cross the PPARG threshold level at which differentiation is irreversibly triggered.
  • Plotted lines are population median traces with shaded regions representing 25th and 75th percentiles of approximately 700 cells per condition, representative of 6 independent experiments.

CONTACT FOR REAGENT AND RESOURCE SHARING

  • Further information and requests for reagents may be directed to, and will be fulfilled by the corresponding author, Dr. Mary N. Teruel (mteruel@stanford.edu).

EXPERIMENTAL MODEL DETAILS

  • Immune-deficient mouse-J:NU (Jackson Labs, Cat #007860).

Cell culture and differentiation

  • OP9 cells were cultured according to previously published protocols [20,23,37,46].
  • Forty-eight hours after initiating differentiation, Stimulus I was removed from the cells and was replaced with Stimulus II consisting of growth media plus 10% FBS and 1.75 nM insulin for another 48 hours.
  • As noted in some experiments, rosiglitazone (Cayman, USA) was added to the media to result in a final concentration of 1 μM. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
  • Page 27 3T3-F442A preadipocytes were grown and differentiated according to established protocols[30].
  • After day 4, the media was replaced with just growth media.

Oil Red O staining

  • To determine lipid accumulation 3T3-F442A preadipocytes were differentiated for 10 days and stained with Oil Red O. Briefly, different guide RNAs targeting FABP4 or FABP5 were designed (crispr.mit.edu) and oligos including the targeting sequences were annealed and cloned into pSpCas9n(BB)-2A-GFP (PX461; Addgene #48140), used for targeting FABP4, or pSpCas9(BB)-2AmiRFP670 (Addgene #91854), used for targeting FABP5.
  • At day 0, the cells were treated with doxycycline 1ug/ml and adipogenic cocktail in MEM with Lglutamine, 10% FBS, and 100 U/ml penicillin/streptomycin.
  • CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
  • Next, western blot analysis of mKate-FABP4 clone2 was performed using anti-RFP antibody and anti-FABP4 to verify protein expression and to check for the correct molecular weight of the tagged protein (Supp Figs S3C and S3D).

Immunoblotting

  • For SDS-PAGE, 20 ug protein per lane was used.
  • Co-Immunoprecipitation (Co-IP) .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
  • Twenty-four hours before the start of the experiment, stable FABP4-KO OP9 cells with either inducible YFP, YFP-WT-FABP4 or YFP-FABP4 (R126Q) were treated with doxycycline (1ug/ml) and in alphaMEM with L-glutamine, 10% FBS, and 100 U/ml penicillin/streptomycin.
  • Co-IP’s on the total cell lysates or on subcellular fractions (nuclear and cytoplasmic) were performed at different timepoints using antiGFP VHH coupled to magnetic agarose beads (GFP-Trap_MA, ) or, as a control, IgG , following the manufacturer’s instructions.
  • The same amount of protein was used for each IP, and the protein concentration was measured using a BSA kit.

FABP4 Gain-of-Function in pre-adipocytes via CRISPRa SAM

  • Murine mesenchymal C3H10T1/2 cells stably expressing the CRISPRa-SAM complex were used for gain of function experiments following a previously established protocol [33].
  • The resulting cell line (C3H10T1/2CRISPRa-SAM) can be used for activation of endogenous genes via chemical transfection with a single guide RNA -containing plasmid.
  • Page 33 experiments, 2000 cells were seeded per well into a 96-well Costar Plastic plate.
  • At Day 2, the media was replaced with media containing only insulin.
  • PCR primer sequences were synthesized by Elim Biopharmaceuticals Inc (USA, CA) and are listed in Supplementary Table S8.

Fluorescent imaging

  • Imaging was conducted using an ImageXpress MicroXL (Molecular Devices, USA) with a 10X Plan Apo 0.45 NA objective.
  • A camera bin of 2x2 was used for all imaging condition.
  • Living cells were imaged in FluoroBrite DMEM media with 10% FBS, 1% Penicillin/Streptomycin and insulin to reduce background fluorescence.
  • Four, non-overlapping sites in each well were imaged.
  • .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Imaging data processing

  • Data processing of fluorescent images was conducted in MATLAB .
  • Unless stated otherwise, fluorescent imaging data were obtained by automated image segmentation, tracking and measurement using the MACKtrack package for MATLAB.
  • If cells were binned according to their PPARG expression, cells were binned based on their mean nuclear PPARG expression at the described timepoints.
  • Briefly, PPARG values at the end of a differentiation experiment typically exhibit a bimodal distribution.
  • Measuring protein decay rates using cycloheximide Protein decay rates were quantified as previously described [20,27].

Statistics

  • Unless specified otherwise, data are expressed as mean +/- standard error of the mean (S.E.M).
  • CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
  • BioRxiv preprint A Adipogenic stimulus (DMI) CEBPB CEBPA FABP4 PPARG.
  • Plotted lines are population median traces with shaded regions representing 25th and 75th percentiles of approximately 700 cells per condition, representative of 6 independent experiments.
  • During Phase 2, both external stimulation and internal self-amplification are needed to increase PPARG levels up to the threshold.

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Bahrami-Nejad,et,al.! Page%1!!
Title: Early enforcement of cell identity by a functional component of the terminally
differentiated state
!
Authors: Zahra Bahrami-Nejad
1,
*
, Tinghuan Chen
1,2,
*
, Stefan Tholen
1
, Zhi-Bo Zhang
1,2
,
Atefeh Rabiee
1
, Michael L. Zhao
1
, Fredric B. Kraemer
3
, and Mary N. Teruel
1,2, #
Affiliations:
1
Dept. of Chemical and Systems Biology, Stanford University, Stanford, CA
2
Dept. of Biochemistry and the Drukier Institute for Children’s Health, Weill Cornell Medicine, New
York, NY
3
Dept. of Medicine/Division of Endocrinology, Stanford University, Stanford, CA and VA Palo Alto
Health Care System, Palo Alto, CA
*
Equal Contribution
!!
#
Corresponding Author: mnt4002@med.cornell.edu
ABSTRACT
How progenitor cells can attain a distinct differentiated cell identity is a challenging problem given that
critical transcription factors are often not unique to a differentiation process and the fluctuating
signaling environment in which cells exist. Here we test the hypothesis that a unique differentiated cell
identity can result from a core component of the differentiated state doubling up as a signaling protein
that also drives differentiation. Using live single-cell imaging in the adipocyte differentiation system,
we show that progenitor fat cells (preadipocytes) can only commit to terminally differentiate after
upregulating FABP4, a lipid buffer that is highly enriched in mature adipocytes. Upon induction of
adipogenesis, we show that after a long delay, cells first abruptly start to engage a positive feedback
between CEBPA and PPARG before then engaging, after a second delay, a positive feedback
between FABP4 and PPARG. These sequential positive feedbacks both need to engage in order to
drive PPARG levels past the threshold for irreversible differentiation. In the last step before
commitment, PPARG transcriptionally increases FABP4 expression while fatty-acid loaded FABP4
binds to and increases PPARG activity. Together, our study suggests a control principle for robust cell
identity whereby a core component of the differentiated state also promotes differentiation from its own
progenitor state.
HIGHLIGHTS
Fatty-acid loaded FABP4 binds to and increases PPARG expression, thereby turning on PPARG
positive feedback loops that further increase PPARG expression.
FABP4 critically controls the second phase of adipogenesis between activation of the feedback
loops and reaching the threshold to differentiate.
Only a small fraction (~10%) of the FABP4 levels typically attained in mature fat cells is needed to
commit cells to the differentiated state, thus providing an explanation for why maintenance of the
mature adipocyte state is so robust.
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted August 14, 2020. ; https://doi.org/10.1101/2020.01.03.894493doi: bioRxiv preprint

Bahrami-Nejad,et,al.! Page%2!!
INTRODUCTION
Terminal cell differentiation is fundamental for developing, maintaining, and regenerating
tissues in all multicellular organisms and is the process by which specialized cells such as adipocytes
(fat cells), osteoblasts, neurons, and muscle cells are generated from progenitor cells [1]. However,
how progenitor cells can reach a specific and unique terminally differentiated state is not well
understood. In many differentiation processes, the critical transcription factors and signaling elements
driving cell fate are not unique to a specific differentiation process [25]. Furthermore, single cell
RNAseq and other single-cell technologies have provided direct evidence that cell-to-cell variability in
protein expression and signaling activities can cause differentiating cells to pass through stochastic
intermediate states that could misdirect cells to alternative final fates [69]. Given the ubiquitous use
of overlapping signaling and transcription programs and the significant signaling variability between
individual cells in a population, this raises the question how a differentiating cell avoids becoming lost
in intermediate states and reliably finds its way to the desired specific terminal cell fate.
As one mechanism supporting terminal cell fate identity, negative feedback has been shown
to play a role in regulating the differentiation decision between multiple fates whereby one fate
suppress the programs that drive differentiation of the other fates [10]. However, since negative
feedback mostly prevents cells which have chosen one path from differentiating into alternative cell
types, additional regulatory mechanisms must exist that selectively drive cells onto a unique path and
allow cells to robustly assume and maintain a specific differentiated cell identity. A number of studies
focusing on directed and transdifferentiation processes showed that differentiation can proceed by
multiple routes and yet converge onto similar transcriptional states [11,12], consistent with the view
that terminally differentiated cell states are ‘attractor basins’ in a transcriptional and signaling
differentiation landscape. The finding that the same differentiated state could be reached by passing
through different intermediate states motivated us to ask the question whether a unique attractor basin
requires a unique cell identify factor that would allow for the same terminally differentiated state to be
reached from different intermediate states. Specifically, we considered that cells may assume a robust
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted August 14, 2020. ; https://doi.org/10.1101/2020.01.03.894493doi: bioRxiv preprint

Bahrami-Nejad,et,al.! Page%3!!
differentiated cell identity by using a type of positive feedback whereby a core component that is
uniquely expressed and needed in the differentiated state doubles-up as a signaling co-factor that
drives an irreversible step in the differentiation process.
Such a self-reinforcing mechanism that promotes robust cell identity (1) would have to control
a late step before commitment to ensure that cells select the correct differentiation path and cell
identity, and (2) once cells have committed, would have to robustly lock the cell into its differentiated
state. To test if such a positive-feedback mechanism exists and how it may reinforce cell identity of a
differentiated state, we used the adipocyte differentiation system since it is a well-characterized and
experimentally accessible terminal cell differentiation process [13]. We were also intrigued by a
previous observation that increased levels of fatty acids can promote differentiation of precursor cells.
As a candidate for such a self-reinforcing mechanism for cell identity, the fatty acid binding protein,
FAPB4, is one of the most abundant proteins in adipocytes, where it makes up between 0.5-6% of
soluble protein [14]. FABP4 is normally expressed at high levels only in adipocytes [15,16] and is a
critical core component of adipocytes that has important cell-internal functions such as binding to
hormone sensitive lipase (HSL) and buffering lipid release [17]. However, there is also evidence that
FABP4 may have an additional role in positively regulating the transition from progenitor cells into
mature adipocytes [1820]. These observations motivated our study here that FABP4 could provide
such a self-reinforcement mechanism for unique cell identity. We note that FABP4 can also be
released from mature adipocytes and has cell-external roles in different cell types including
preadipocytes and other adipocytes where it has been shown to reduce PPARG expression as a
pathological and possibly also normal regulatory function [21].
It is well-established that the expression of FABP4 is induced by PPARG, the master regulator
of adipocyte differentiation [22]. However, whether FABP4 has a role in regulating PPARG activity
during the differentiation process has been difficult to resolve since very little FABP4 is expressed
early in adipogenesis [16,23] and that cells in the population differentiate at different times making it
challenging to use traditional bulk cell assays. Also, it is not clear how FABP4 regulates PPARG and
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted August 14, 2020. ; https://doi.org/10.1101/2020.01.03.894493doi: bioRxiv preprint

Bahrami-Nejad,et,al.! Page%4!!
if and how FABP4 and PPARG may reinforce each other’s expression. Finally, if FABP4 is indeed
uniquely important for adipogenesis, it is not clear why genetic studies in FABP4-knockout mice failed
to show a suppression of adipogenesis [24].
Here, using live single-cell analysis of fluorescently-tagged endogenous PPARG and FABP4,
we show that FABP4 and PPARG build up only very slowly during a first phase of the adipogenic
program until they transition to a second phase marked by engagement of positive feedback between
each other. This feedback starts after about 24 hours when the FABP4 level is very low and ends
approximately 12 hours later when the levels of PPARG and FABP4 rapidly build up and pass a critical
threshold for differentiation. We show that the feedback-activation of PPARG can be repressed by a
mutant FABP4 that is deficient in binding fatty acid and that FABP4 can directly interact with PPARG.
This suggests that FABP4 has - at much lower levels than seen in differentiated cells - a transport
function to enhance fatty acid binding to PPARG, a mechanism which is known to increase PPARG
activity [22]. Finally, we show that FABP5 may compensate for a loss of FABP4 and still allow cells to
differentiate. Together, our study provides support for a likely more general model that robust cell
identity can be initiated and reinforced by having a unique core component of the differentiated state
double-up as a signaling factor that initiates and then reinforces the path to this unique differentiated
state.
RESULTS
FABP4 is needed for adipogenesis in vitro and in vivo
Previous studies suggested that FABP4 and PPARG are in a positive feedback relationship
[18,20,23,25] (Fig 1A). One arm of this positive feedback is well-established since there are PPARG
binding sites on the FABP4 promoter, and PPARG activity has been shown to strongly upregulate
FABP4 mRNA and protein levels [14]. However, the relevance of FABP4 in regulating PPARG has
been controversial as FABP4 knockout mice are not defective in adipogenesis [24]. We therefore
performed a series of experiments to address if and how FABP4 can regulate PPARG expression and
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted August 14, 2020. ; https://doi.org/10.1101/2020.01.03.894493doi: bioRxiv preprint

Bahrami-Nejad,et,al.! Page%5!!
adipogenesis. We first carried out CRISPR-mediated genome editing to completely knock out FABP4
expression in OP9 cells (Fig 1B). We then induced adipogenesis using the standard 96-hour DMI
protocol and measured the percent of differentiated cells using a previously established single-cell
immunocytochemistry assay based on assessing whether PPARG expression is above or below a
threshold [20,23,26,27] (see Methods). As expected, control-KO OP9 cells differentiated robustly (Fig
1C). In contrast, FABP4-KO OP9 cells were strongly defective in increasing PPARG expression and
differentiating, with less than 10% of cells differentiating compared to control cells. Since FABP5 has
been shown to compensate for FABP4 knock-out both in vitro and in vivo [24,28,29], we also tested
whether we could further reduce the amount of adipogenesis in the FABP4-KO cells by also knocking
down FABP5 using siRNA. Indeed, we could reduce adipogenesis to almost nothing when we knocked
down both FABP4 and FABP5 (Fig 1C).
We further validated these findings in another commonly used preadipocyte cell system, 3T3-
F442A cells [30]. We used CRISPR-mediated genome editing to knockout FABP4 or both FABP4 and
FABP5 (Fig 1D). 3T3-F442A cells which had been subjected to the same FABP4 knockout protocol,
but which did not harbor any FABP4 knockout were used as control cells. We plated the 3T3-F442A
cells into 96-well plates and induced adipogenesis using the standard protocol in 3T3-F442A cells
which is to add insulin[30]. Indeed, when FABP4 was knocked out, 3T3-F442A preadipocytes showed
reduced differentiation, as measured by PPARG staining, as well as lipid accumulation, as measured
by Oil Red-O staining (Fig 1E and 1F). The latter is indicative that the KO cells are not functional
mature adipocytes. To test whether FABP4 was essential for adipogenesis in vivo, we used a
previously-established method in which 3T3-F442A preadipocytes are subcutaneously injected into
the sternum of immune-deficient mice, giving rise to fat pads resembling normal adipose tissue[31].
Since fat is not normally present at the sternum of mice, the fat pad formed at the sternum after
injection of preadipocyte cells is generated by de novo adipogenesis of the injected cells[31]. We
injected our preadipocyte cells into the sternum of 8-week mice, and a fat pad was allowed to form for
4 weeks. The FABP4-KO and FABP4/FABP5 double knockout (DBKO) preadipocyte cells indeed
.CC-BY-NC-ND 4.0 International licenseavailable under a
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The copyright holder for this preprintthis version posted August 14, 2020. ; https://doi.org/10.1101/2020.01.03.894493doi: bioRxiv preprint

Citations
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01 Jan 1997-
TL;DR: These findings indicate that adipose-specific promoter-reporter constructs, transfected into 3T3-F442A preadipocytes, can be tested in an in vivo context during and after development of these cells into adipose tissue, and offer a faster and less costly alternative to the transgenic mouse method for assessing adipose gene function.
Abstract: T3-F442Apreadipocytesimplanteds.c.into athymic mice develop into fat pads that are indistinguishable fromnormaladiposetissue.Implantedpreadipocytesharbor- ing a b-galactosidase transgene gave rise to fat pads in which almost all adipocytes expressed b-galactosidase. This finding proved that the implanted 3T3-F442A preadipocytes, rather than endogenous preadipose cells, gave rise to the newly developed ''adipose tissue.'' 3T3-F442A preadipocytes, when differentiatedintoadipocytesincellculture,expresstheobese geneatanunexpectedlylowlevel,i.e.,<1%thelevelinadipose tissue. However, adipose tissue derived from s.c. implanted 3T3-F442A preadipocytes expressed leptin mRNA at a level comparable to that in epididymal adipose tissue. These find- ingsindicatethatafactor(s)orcondition,presentinthetissue context and necessary for maximal obese gene expression, is lacking in cell culture. Furthermore, adipocytes derived from the implanted cells were hormonally responsive in that leptin mRNA levels were up-regulated 3- to 8-fold by glucocorticoid injection into the host animal. Thus, these findings indicate that adipose-specific promoter-reporter constructs, trans- fected into 3T3-F442A preadipocytes, can be tested in an in vivocontext during and after development of these cells into adipose tissue. Furthermore, the effect of transgenes on the adipogenicdevelopmentoftheimplantedpreadipocytescanbe assessed. Thus, this approach offers a faster and less costly alternative to the transgenic mouse method for assessing adipose gene function.

6 citations


Posted ContentDOI
21 Apr 2020-bioRxiv
TL;DR: A rapid switch mechanism engages exclusively in G1 to trigger a simultaneous commitment to differentiate and permanently exit from the cell cycle and the differentiation control system is able to couple mitogen and differentiation stimuli to sustain a long-term balance between terminally differentiating cells and maintaining the progenitor cell pool.
Abstract: Terminal differentiation is essential for the development and maintenance of tissues in all multi-cellular organisms and is associated with a permanent exit from the cell cycle. Failure to permanently exit the cell cycle can result in cancer and disease. However, the molecular mechanisms and timing that coordinates differentiation commitment and cell cycle exit are not yet understood. Here using adipogenesis as a model system to track differentiation commitment in live cells, we show that a rapid switch mechanism engages exclusively in G1 to trigger a simultaneous commitment to differentiate and permanently exit from the cell cycle. We identify a signal integration mechanism whereby the strengths of both mitogen and differentiation stimuli control a molecular competition between cyclin D1 and PPARG-induced expression of the CDK inhibitor p21 which in turn regulates if and when the differentiation switch is triggered and when the proliferative window closes. In this way, the differentiation control system is able to couple mitogen and differentiation stimuli to sustain a long-term balance between terminally differentiating cells and maintaining the progenitor cell pool, a parameter of critical importance for enabling proper development of tissue domains and organs.

2 citations


Journal Article
TL;DR: Functional characterization of C/EBPa and C/ EBPb proteomes suggests they can regulate novel pathways and indicate potential molecular targets for therapeutic intervention.
Abstract: C/EBPa and C/EBPb are transcription factors with tissue specific expression regulating several important cellular processes. They work by recruiting protein complexes to a common DNA recognition motif and both are able to compensate each other's absence in many cell types, thus showing functional redundancy. They also play distinct roles in specific cellular pathways and their abnormal functioning gives raise to different human pathologies.To investigate the molecular basis of C/EBPa and C/EBPb specificity and redundancy we characterized their in vivo protein-protein interaction networks by Tandem Affinity Purification (TAP) and Mass Spectrometry (MS). To unravel the functional features of C/EBPa and C/EBPb proteomes we studied the statistical enrichment of binding partners related to Gene Ontology (GO) terms and KEGG pathways.Our data confirmed that the C/EBPa and C/EBPb regulate biological processes like cell proliferation, apoptosis and transformation. We found that both C/EBPa and C/EBPb are involved in other cellular pathways such as RNA maturation, RNA splicing and DNA repair. Specific interactions of C/EBPa with MRE11, RUVBL1 and RUVBL2 components of DNA repair system were confirmed by co-immunoprecipitation assays.Our comparative analysis of the C/EBPa and C/EBPb proteomes provides an insight for understanding both their redundant and specific roles in cells indicating their involvement in new pathways. Such novel predicted functions are relevant to normal cellular processes and disease phenotypes controlled by these transcription factors.Functional characterization of C/EBPa and C/EBPb proteomes suggests they can regulate novel pathways and indicate potential molecular targets for therapeutic intervention.

1 citations


Journal ArticleDOI
Md. Shamim Rahman1, Inseok Kang1, Youri Lee1, Md. Ahasun Habib1  +4 moreInstitutions (2)
Abstract: Although the health benefits of probiotics have been widely known for decades, there has still been limited use of probiotic bacteria in anti-obesity therapy. Herein, we demonstrated the role of Bifidobacterium longum subsp. infantis YB0411 (YB, which was selected by an in vitro adipogenesis assay) in adipogenic differentiation in 3T3-L1 pre-adipocytes. We observed that YB-treatment effectively reduced triglyceride accumulation and the expression of CCAAT/enhancer-binding protein α, β, and δ (C/EBPα, C/EBPβ, and C/EBPδ), peroxisome proliferator-activated receptor γ (PPARγ), fatty acid-binding protein 4 (aP2), and acetyl-CoA carboxylase (ACC). YB-treatment also reduced the levels of core autophagic markers (p62 and LC3B) in 3T3-L1 pre-adipocytes. Small-interfering-RNA-mediated knockdown and competitive-chemical-inhibition assays showed that AMP-activated protein kinase (AMPK) commenced the anti-adipogenic effect of YB. In addition, YB supplement markedly reduced body weight and fat accretion in mice with high-fat-diet-induced obesity. Our findings suggest that YB may be used as a potential probiotic candidate to ameliorate obesity.

References
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Journal ArticleDOI
23 Jan 2015-Science
Abstract: Resolving the molecular details of proteome variation in the different tissues and organs of the human body will greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on an integrated omics approach that involves quantitative transcriptomics at the tissue and organ level, combined with tissue microarray-based immunohistochemistry, to achieve spatial localization of proteins down to the single-cell level. Our tissue-based analysis detected more than 90% of the putative protein-coding genes. We used this approach to explore the human secretome, the membrane proteome, the druggable proteome, the cancer proteome, and the metabolic functions in 32 different tissues and organs. All the data are integrated in an interactive Web-based database that allows exploration of individual proteins, as well as navigation of global expression patterns, in all major tissues and organs in the human body.

6,953 citations


Journal ArticleDOI
F. Ann Ran, Patrick D. Hsu, Jason Wright1, Vineeta Agarwala1  +3 moreInstitutions (2)
01 Nov 2013-Nature Protocols
TL;DR: A set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies are described.
Abstract: Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.

6,858 citations


"Early enforcement of cell identity ..." refers methods in this paper

  • ...Generation of FABP4-KO, FABP5-KO, and DBKO cells: The CRISPR-Cas9 constructs targeting FABP4 and FABP5 were generated based on a previously described protocol [39]....

    [...]


Journal ArticleDOI
30 Dec 1994-Cell
TL;DR: The results suggest that the physiologic role of PPAR gamma 2 is to regulate development of the adipose lineage in response to endogenous lipid activators and that this factor may serve to link the process of adipocyte differentiation to systemic lipid metabolism.
Abstract: Peroxisome proliferator-activated receptor gamma 2 (PPAR gamma 2) is an adipocyte-specific nuclear hormone receptor that has recently been identified as a key regulator of two fat cell enhancers. Transcriptional activation by PPAR gamma 2 is potentiated by a variety of lipids and lipid-like compounds, including naturally occurring polyunsaturated fatty acids. We demonstrate here that retroviral expression of PPAR gamma 2 stimulates adipose differentiation of cultured fibroblasts. PPAR activators promote the differentiation of PPAR gamma 2-expressing cells in a dose-dependent manner. C/EBP alpha, a second transcription factor induced during adipocyte differentiation, can cooperate with PPAR gamma 2 to stimulate the adipocyte program dramatically. Our results suggest that the physiologic role of PPAR gamma 2 is to regulate development of the adipose lineage in response to endogenous lipid activators and that this factor may serve to link the process of adipocyte differentiation to systemic lipid metabolism.

3,313 citations


Journal ArticleDOI
12 Sep 2013-Cell
Abstract: Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.

2,663 citations


01 Sep 2013-
TL;DR: It is demonstrated that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency.
Abstract: Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.

1,940 citations


"Early enforcement of cell identity ..." refers methods in this paper

  • ...Workflow used to generate single cell colonies with endogenously tagged PPARG and FABP4 (mouse OP9 cells, Fig S3): To carry out CRISPR genome editing to generate FABP4-mkate2, we used the “double nickase” system which uses two different guide RNAs that create adjacent and opposing nicks in the DNA at the site of insertion [11,41]....

    [...]


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