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Puromycin Selection Confounds the RNA-Seq Profiles of Primary Human Erythroblasts

January 2017

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National Institutes of Health

Flow cytometry profiles. Representative flow dot plots of control cells compared to cells transduced with either SHC002V or CLS-NCG-8 analyzed on culture day 14.

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Venn diagram comparisons. Venn diagrams comparing the gene profiles of control (blue), SHC002V (red), and CLS-NCG-8 (yellow). (A) and (B) compare the control with each transduced sample. Genes in the blue area were significantly down-regulated by the lentivirus, genes in the red or yellow area were significantly up-regulated, and genes in the overlapped area were insignificantly differentially expressed. (C) compares the two transduced groups with each other (SHC002V vs. CLS-NCG-8).

…

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Volume 5 • Issue 1 • 1000140

Transcriptomics, an open access journal

ISSN: 2329-8936

Transcriptomics: Open Access

Guo et al., Transcriptomics 2017, 5:1

DOI: 10.4172/2329-8936.1000140

Short Communication OMICS International

Puromycin Selection Confounds the RNA-Seq Profiles of Primary Human

Erythroblasts

Guo RL, Lee YT, Byrnes C, and Miller JL*

Molecular Genomics and Therapeutics Section, Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National

Institutes of Health, Bethesda, Maryland, USA

Abstract

Lentiviral transduction followed by puromycin selection is a well-recognized procedure for gene transfer and

expression experiments using a variety of cell types including human hematopoietic stem and progenitor cells. Despite its

widespread application, research regarding the potential effects of bacterial puromycin N-acetyltransferase (pac) gene

expression in mammalian cell cultures is incomplete. Here the potential for puromycin selection to affect transcriptome

proles was examined using a well-studied model for human erythropoiesis. Experiments were performed using

primary CD34(+) cells from six adult healthy human donors transduced with two commercially available pac-encoding

lentiviral vectors and compared to non-transduced control cells. RNA-Seq gene expression proles were generated at

the proerythroblast stage of differentiation, then differential gene expression was analyzed with DEseq2 in R-Studio

software. Inter-donor variation in the gene expression proles and variations between puromycin selected populations

after transduction of the separate lentiviral vectors was manifested by signicant differences in the RNA detection

levels of less than 0.1%. However, puromycin selection after pac gene transduction caused signicant changes in over

5% of the mRNA when compared to non-transduced controls. The results suggest that consideration should be given

for the potential of puromycin selection to confound the interpretation of RNA-Seq transcriptome proles.

*Corresponding author: Jeffery L Miller, Genetics of Development and Disease

Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National

Institutes of Health, 10 Center Drive, Building 10, Room 9N311, Bethesda,

Maryland 20892. USA, Tel: 3014801908; E-mail: jm7f@nih.gov

Received April 01, 2017; Accepted April 27, 2017; Published May 01, 2017

Citation: Guo RL, Lee YT, Byrnes C, Miller JL (2017) Puromycin Selection

Confounds the RNA-Seq Proles of Primary Human Erythroblasts. Transcriptomics

5: 140. doi:10.4172/2329-8936.1000140

the terms of the Creative Commons Attribution License, which permits unrestricted

use, distribution, and reproduction in any medium, provided the original author and

source are credited.

Keywords: Puromycin; Erythropoiesis; Drug selection; Gene

transfer; RNA-seq

Short Communication

Puromycin, an amino nucleoside antibiotic, is biosynthesized by the

bacterium species Streptomyces alboniger. e biomolecular structure

of puromycin resembles that of an aminoacyl-tRNA molecule. Its main

mechanism of action involves blocking protein synthesis through the

interference with the peptidyl transfer procedure during translation

[1,2]. Its amino group disrupts the ester bond between the nascent

peptide and tRNA, allowing for the puromycin molecule to attach itself

to the C-terminus. It contains an amide bond instead of ester bond, thus

preventing the next aminoacyl-tRNA from appending itself. Premature

chain termination thus occurs, and protein synthesis is aborted [3].

e pac gene encodes puromycin N-acetyltransferase, thus

conferring puromycin resistance [4]. Puromycin N-acetyltransferase

acetylates the nitrogen atom of the amino group in the puromycin

tyrosinyl moiety, thus preventing it from breaking the ester bond in

the peptidyl-tRNA. Since the puromycin molecule is no longer able to

attach itself to the peptide chain, it becomes biologically inactive, and

protein synthesis continues [2].

Predictable resistance to puromycin-mediated cell death aer

pac gene transduction was identied as a selection strategy for

gene manipulation in eukaryotic cells [5]. More recently, lentiviral

transduction followed by puromycin resistance has become an

established procedure for exploring the eects of transgene expression

in a variety of model systems using high-throughput RNA sequencing

(RNA-Seq) with hematopoietic stem and progenitor cells [6-8]. Despite

utilization of this experimental approach, the potential eect of the

bacterial puromycin N-acetyltransferase gene upon the RNA-Seq

proles remains vague.

In this study, we explored the potential eects of puromycin

selection upon RNA-Seq proles using cultured primary hematopoietic

cells. Human CD34(+) cells were cultured ex vivo from healthy adult

human donors as previously described in a 3-week serum free culture

system [9]. Prior to this study, written informed consent was obtained

from research subjects. Consent documents regarding studies using

primary erythroblasts and approval of the research protocol were

obtained from the National Institute of Diabetes and Digestive and

Kidney DiseasesInstitutional Review Board.

Figure 1: Puromycin titration. The percent of live cells at (A) culture day 7 and

(B) culture day 14.

Citation: Guo RL, Lee YT, Byrnes C, Miller JL (2017) Puromycin Selection Confounds the RNA-Seq Proles of Primary Human Erythroblasts.

Transcriptomics 5: 140. doi:10.4172/2329-8936.1000140

Page 2 of 3

Volume 5 • Issue 1 • 1000140

Transcriptomics, an open access journal

ISSN: 2329-8936

An initial puromycin titration experiment was performed on cells

from three donors using 0.1 µg/ml to 1.0 µg/ml dosage to determine

the killing concentration aer 3-day selection (puromycin selection on

culture days 4-6). Puromycin was purchased from Sigma Aldrich (St.

Louis, MO). At culture day 7, ow cytometry analysis was performed to

determine the percent of live cells with a total of 10,000 events recorded

using a BD FACSAria I ow cytometer (BD Biosciences, San Jose, CA).

e cells were then transferred to phase II media without puromycin

for all conditions for an additional 7 days. On culture day 14, ow

cytometry was again performed to determine if any cells survived the

puromycin selection. e most eective concentration of puromycin

for 3-day selection was found to be 0.7 µg/ml based on less than 2% live

cells on culture day 7 and no surviving cells on culture day 14 from the

average of three donors (Figure 1).

For puromycin selection experiments, lentiviral transduction

was performed on cells from six donors as previously described [10].

Briey, the cells were transduced on day 3 of culture with either

SHC002V (Sigma Aldrich) or CLS-NCG-8 (Qiagen, Valencia, CA).

e next day, puromycin was added at 0.7 µg/ml for an additional 3

days. At culture day 7, the cells were transferred to phase II media

without puromycin. On culture day 14, erythroblast dierentiation

was assessed using ow cytometry analysis with the BD FACSAria I

ow cytometer to determine if maturation of the transduced cells was

aected as compared to control. e cells were stained with antibodies

directed against transferrin receptor, CD71 (Invitrogen, Carlsbad, CA)

and glycophorin A (Invitrogen). ere was no observed dierence in

cell dierentiation between the control and transduced cells (Figure 2).

To explore potential eects of puromycin selection on the RNA-Seq

proles of human primary erythroblasts, live cells from six healthy adult

human donors were sorted on culture day 14, and RNA was extracted

using the miRNeasy mini kit (Qiagen). Globin messages were depleted

from 1.0 μg of total RNA using the GLOBINclear Human Kit and rRNA

was depleted using the Ribo-Zero Gold rRNA Removal Kit (Human/

Mouse/Rat). Aer polyA-selection and depletion, the total RNA was

used for cDNA library generation. An Illumina HiSeq 2000 (version

3 chemistry) was utilized to sequence the pooled libraries on multiple

lanes. At least 40 million 101 base pair reads were achieved.

e Illumina sequence reads were aligned against human genome

build hg19 via Illumina sequencing soware, Real-Time Analysis

version 1.13.48, CASAVA version 1.8.2 and Ecient Large-Scale

Alignment of Nucleotide Databases (ELAND) mapping algorithm. e

separate lane reads were merged into BAM les, which were loaded

into the SamtoFastq tool to generate FASTQ les. Each FASTQ le was

processed with FASTQ-Trimmer module and FASTQ-Masker module

of FASTX-Toolkit (version 0.0.14) to trim 21 base pair from the end

and mask any sequence base with a quality score less than 30 with an

N to generate a nal 80 base pair FASTQ le for aligning against the

Human Genome hg19 build with Spliced Transcripts Alignment to a

Figure 2: Flow cytometry proles. Representative ow dot plots of control cells compared to cells transduced with either SHC002V or CLS-NCG-8 analyzed on culture

day 14.

Figure 3: Venn diagram comparisons. Venn diagrams comparing the gene proles of control (blue), SHC002V (red), and CLS-NCG-8 (yellow). (A) and (B) compare

the control with each transduced sample. Genes in the blue area were signicantly down-regulated by the lentivirus, genes in the red or yellow area were signicantly

up-regulated, and genes in the overlapped area were insignicantly differentially expressed. (C) compares the two transduced groups with each other (SHC002V vs.

CLS-NCG-8).

Comparison No. of Genes Insignicantly Differentially

Expressed (FDR-adjusted p-value >0.01)

No. of Genes Signicantly Differentially

Expressed (FDR-adjusted p-value <0.01)

No. Down-

regulated

No. Up-

regulated

Between Conditions Control versus SHC002V 24803 1560 898 662

Control versus CLS-NCG-8 24949 1414 811 603

SHC002V versus CLS-NCG-8 26358 5 3 2

Inter-donor Variation Control versus Control 26359 42 2

SHC002V versus SHC002V 26363 0 0 0

CLS-NCG-8 versus CLS-NCG-8 26361 2 1 1

Table 1: Comparison of RNA-Seq identied genes.

Citation: Guo RL, Lee YT, Byrnes C, Miller JL (2017) Puromycin Selection Confounds the RNA-Seq Proles of Primary Human Erythroblasts.

Transcriptomics 5: 140. doi:10.4172/2329-8936.1000140

Page 3 of 3

Volume 5 • Issue 1 • 1000140

Transcriptomics, an open access journal

ISSN: 2329-8936

other primary tissues or cancer cell lines. In this preliminary study,

mechanisms responsible for the dramatic change in the transcriptome

proles were not identied. We speculate that lentiviral transduction

of the pac gene may have o target eects that indirectly aect RNA

transcription or stability. Also, puromycin itself may have eects on

the cells that are not reversed by pac gene expression. Importantly,

analogous eects from puromycin selection have been reported

previously [5]. Other reports suggest that lentiviral transduction

conferring puromycin resistance may lead to a misfolded protein

response in human cell lines [12]. us, adequate controls should be

incorporated in experimental designs in other model systems to more

clearly interpret or compare high-throughput gene expression proles

in puromycin selected cells.

References

1. De la Luna S, Ortín J (1992) [33] pac gene as efcient dominant marker and

reporter gene in mammalian cells. Methods Enzymol. 216: 376-385.

2. Vara J, Perez-Gonzalez JA, Jimenez A (1985) Biosynthesis of puromycin by

Streptomyces alboniger: Characterization of puromycin N-acetyltransferase.

Biochemistry 27: 8074-8081.

3. Palmer M, Chan A, Dieckmann T, Honek J (2012) Biochemical Pharmacology.

John Wiley & Sons, Inc., Hoboken 271-273.

4. Lacalle RA, Pulido D, Vara J, Zaiacaín M, Jiménez A (1989) Molecular analysis

of the pac gene encoding a puromycin N-acetyl transferase from Streptomyces

alboniger. Gene 2: 375-380.

5. Lanza AM, Kim DS, Alper HS (2013) Evaluating the inuence of selection

markers on obtaining selected pools and stable cell lines in human cells.

Biotechnol J 7: 811-821.

6. Lee YT, de Vasconcellos JF, Yuan J, Byrnes C, Noh S, et al. (2013)

LIN28B-mediated expression of fetal hemoglobin and production of fetal-like

erythrocytes from adult human erythroblasts ex vivo. Blood 6:1034-1041.

7. Ali N, Karlsson C, Aspling M, Hu G, Hacohen N, et al. (2009) Forward RNAi

screens in primary human hematopoietic stem/progenitor cells. Blood 16:

3690-3695.

8. Sims D, Mendes-Pereira AM, Frankum J, Burgess D, Cerone MA (2011) High-

throughput RNA interference screening using pooled shRNA libraries and next

generation sequencing. Genome Biology 10: 104.

9. de Vasconcellos JF, Lee YT, Byrnes C, Tumburu L, Miller JL (2016)

HMGA2 moderately increases fetal hemoglobin expression in human adult

erythroblasts. PLoS ONE 11: 0166928.

10. Lee YT, de Vasconcellos JF, Byrnes C, Kaushal M, Miller JL (2015) Erythroid-

specic expression of LIN28A is sufcient for robust gamma-globin gene and

protein expression in adult erythroblasts. PLoS ONE 12:0144977.

11. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C (2013) STAR: Ultrafast

universal RNA-seq aligner. Bioinformatics 29: 15-21.

12. Moran DM, Shen H, Maki CG (2009) Puromycin-based vectors promote a

ROS-dependent recruitment of PML to nuclear inclusions enriched with HSP70

and Proteasomes. BMC Cell Biology 10: 32.

Reference (STAR) soware [11]. Quantitation of 26,363 named gene

transcripts was performed using HTseq. Dierential gene expression

analysis was performed with DESeq2 based on applying negative

binomial generalized linear models in R package and R-Studio to

compare the expression levels of each transcript between SHC002V,

CLS-NCG-8, and control samples. Aer applying the Benjamini-

Hochberg procedure, genes were considered signicantly up- or down-

regulated if the adjusted p-value of the test statistics was less than the

false discovery rate (FDR<0.01) with log2 of ≥1 for up-regulated or

≤-1 for down-regulated genes and baseMean value above 25. e same

bioinformatics procedure was then repeated to quantify the eects of

inter-donor variation by comparing multiple donors within SHC002V,

CLS-NCG-8, and control. RNA-Seq Bam les from each experiment

were deposited in Gene Expression Omnibus (GEO, https://www.

ncbi.nlm.nih.gov/geo/) and released to the public: SRP097005 (Adult

CD34+), SRP097630 (Cord CD34+), SRP096196 (CLS-NCG-8), and

SRP098089 (SHC002V).

e results showed that SHC002V signicantly aected expression

of 1560 genes (898 down-regulated, 662 up-regulated), comprising

approximately 5.9% of the identied transcripts. Whereas, CLS-

NCG-8 had a total of 1414 signicantly dierentially expressed genes

(representing approximately 5.4% of the total prole), with 811 down-

regulated and 603 up-regulated. Each of these vectors has a dierent

recombinant lentivirus encoding the puromycin N-acetyltransferase

gene, a 600-nucleotide fragment originating from Lactobacillus

harbinensis. Consequently, comparisons between SHC002V and CLS-

NCG-8 revealed only 5 genes to be signicantly dierentially expressed

(less than 0.1% of the total gene IDs), suggesting that both lentiviruses

had similar eects on the transcriptome proles of the samples.

To determine the eects of inter-donor variation upon the RNA-

Seq proles, donors within the same condition were compared. e

inter-donor variation tests between the control, SHC002V, and CLS-

NCG-8 revealed only 4, 0, and 2 signicantly dierentially expressed

genes, respectively, which is less than 0.1% of the total number of genes

identied. us, neither dierences in cell maturation, nor inter-donor

variation in RNA-Seq proles likely caused the signicant dierences

between the gene proles of puromycin selected and non-transduced

samples (Table 1 and Figure 3).

ese novel and unexpected results suggest that puromycin

selection may cause broad and signicant changes in the RNA-Seq

proles of cultured primary hematopoietic cells. erefore, selection

aer expression of puromycin N-acetyltransferase can potentially

be a confounding variable for experimental design, interpretation,

and comparison of high-throughput sequencing data. While our

experiments were limited to eects in primary human erythroblasts,

similar comparisons should be considered in alternate models including

Citation:

Guo RL, Lee YT, Byrnes C, Miller JL (2017) Puromycin Selection

Confounds the RNA-Seq Proles of Primary Human Erythroblasts.

Transcriptomics 5: 140. doi:10.4172/2329-8936.1000140

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RNA interference (RNAi) screening is a state-of-the-art technology that enables the dissection of biological processes and disease-related phenotypes. The commercial availability of genome-wide, short hairpin RNA (shRNA) libraries has fueled interest in this area but the generation and analysis of these complex data remain a challenge. Here, we describe complete experimental protocols and novel open source computational methodologies, shALIGN and shRNAseq, that allow RNAi screens to be rapidly deconvoluted using next generation sequencing. Our computational pipeline offers efficient screen analysis and the flexibility and scalability to quickly incorporate future developments in shRNA library technology.

Puromycin-based vectors promote a ROS-dependent recruitment of PML to nuclear inclusions enriched with HSP70 and Proteasomes

Article

Full-text available

Feb 2009
BMC CELL BIOL

Promyelocytic Leukemia (PML) protein can interact with a multitude of cellular factors and has been implicated in the regulation of various processes, including protein sequestration, cell cycle regulation and DNA damage responses. Previous studies reported that misfolded proteins or proteins containing polyglutamine tracts form aggregates with PML, chaperones, and components of the proteasome, supporting a role for PML in misfolded protein degradation. In the current study, we have identified a reactive oxygen species (ROS) dependent aggregation of PML, small ubiquitin-like modifier 1 (SUMO-1), heat shock protein 70 (HSP70) and 20S proteasomes in human cell lines that have been transiently transfected with vectors expressing the puromycin resistance gene, puromycin n-acetyl transferase (pac). Immunofluorescent studies demonstrated that PML, SUMO-1, HSP70 and 20S proteasomes aggregated to form nuclear inclusions in multiple cell lines transfected with vectors expressing puromycin (puro) resistance in regions distinct from nucleoli. This effect does not occur in cells transfected with identical vectors expressing other antibiotic resistance genes or with vectors from which the pac sequence has been deleted. Furthermore, ROS scavengers were shown to ablate the effect of puro vectors on protein aggregation in transfected cells demonstrating a dependency of this effect on the redox state of transfected cells. Taken together we propose that puromycin vectors may elicit an unexpected misfolded protein response, associated with the formation of nuclear aggresome like structures in human cell lines. This effect has broad implications for cellular behavior and experimental design.

Forward RNAi screens in primary human hematopoietic stem/progenitor cells

Article

Full-text available

Mar 2009
BLOOD

The mechanisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood. We report here a screening strategy developed to assess modulators of human hematopoiesis using a lentiviral short hairpin RNA (shRNA) library transduced into cord blood-derived stem/progenitor cells. To screen for modifiers of self-renewal/differentiation, we used the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs conferring enhanced maintenance or expansion of the stem/progenitor potential. This approach enables complex, pooled screens in large numbers of cells. Functional selection identified novel specific gene targets (exostoses 1) or shRNA constructs capable of altering human hematopoietic progenitor differentiation or stem cell expansion, respectively, thereby demonstrating the potential of this forward screening approach in primary human stem cell populations.

Evaluating the Influence of Selection Markers on Obtaining Selected Pools and Stable Cell Lines in Human Cells.

Article

Jul 2013
Biotechnol J

Selection markers are common genetic elements used in recombinant cell line development. While several selection systems exist for use in mammalian cell lines, no previous study has comprehensively evaluated their performance in the isolation of recombinant populations and cell lines. Here we examine four antibiotics, hygromycin, neomycin, puromycin, and Zeocin, and their corresponding selector genes, using a green fluorescent protein (GFP) as a reporter in two model cell lines, HT1080 and HEK293. We identify Zeocin as the best selection agent for cell line development in human cells. In comparison to the other selection systems, Zeocin is able to identify populations with higher fluorescence levels, which in turn leads to the isolation of better clonal populations and less false positives. Further, Zeocin-resistant populations exhibit better transgene stability in the absence of selection pressure compared to other selection agents. All isolated Zeocin-resistant clones, regardless of cell type, exhibited GFP expression. By comparison, only 79% of hygromycin-resistant, 47% of neomycin-resistant and 14% of puromycin-resistant clones expressed GFP. Based on these results, we would rank Zeocin > hygromycin ∼ puromycin > neomycin for cell line development in human cells. Furthermore, this study demonstrates that selection marker choice does impact cell line development.

Molecular analysis of the pac gene encoding a puromycin N-acetyl transferase from Streptomyces alboniger

Article

Aug 1989
GENE

Nucleotide sequence of a 906-bp fragment of Streptomyces alboniger DNA containing the gene (pac), which encodes a puromycin N-acetyltransferase (PAC), has been determined. The pac gene contains a 600-nt open reading frame, starting with an ATG codon, which encodes a polypeptide of Mr 21,531; this is consistent with the 23 +/- 1.5 kDa size of the PAC enzyme. High-resolution S1 mapping indicates that transcription starts at or next to a C residue 35 bp upstream from the putative ATG start codon. A 263-bp DNA fragment from the 5' region of the pac gene has promoter activity in the promoter-probe plasmid pIJ486. Its -35 and -10 regions show significant structural homology to the corresponding regions of the hyg gene promoter, but they are different from the promoter sequences of other Streptomyces and Escherichia coli genes.

Biosynthesis of puromycin by Streptomyces alboniger. Characterization of puromycin N-acetyltransferase

Article

Jan 1986

Puromycin N-acetyltransferase from Streptomyces alboniger inactivates puromycin by acetylating the amino position of its tyrosinyl moiety. This enzyme has been partially purified by column chromatography through DEAE-cellulose and Affigel Blue and characterized. It has an Mr of 23 000, as determined by gel filtration. In addition to puromycin, the enzyme N-acetylates O-demethylpuromycin, a toxic precursor of the antibiotic, and chryscandin, a puromycin analogue antibiotic. The Km values for puromycin and O-demethylpuromycin are 1.7 and 4.6 microM, respectively. The O-demethylpuromycin O-methyltransferase from S. alboniger, which apparently catalyzes the last step in the biosynthesis of puromycin [Rao, M. M., Rebello, P. F., & Pogell, B. M. (1969) J. Biol. Chem. 244, 112-118], also O-methylates N-acetyl-O-demethylpuromycin. The Km values of the methylating enzyme for O-demethylpuromycin and N-acetyl-O-demethylpuromycin are 260 and 2.3 microM, respectively. These findings suggest that O-demethylpuromycin, if present in S. alboniger, would be N-acetylated and then O-methylated to be converted into N-acetylpuromycin. It might even be possible that N-acetylation of the puromycin backbone takes place at an earlier precursor.

Puromycin Selection Confounds the RNA-Seq Profiles of Primary Human Erythroblasts

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