Regulation of RHOV signaling by interaction with SH3 domain-containing adaptor proteins and phosphorylation by PKA.

RHOV and RHOU are considered atypical Rho-family small GTPases because of the existence of N- and C-terminal extension regions, abnormal GDP/GTP cycling, and post-translational modification. Particularly, RHOV and RHOU both have a proline-rich (PR) motif in the N-terminal region. It has been reported that the PR motif of RHOU interacts with GRB2, a SH3 domain-containing adaptor protein, and regulates its activity through EGF receptor signaling. However, it is unknown whether RHOV, like RHOU, interacts with SH3 domain-containing adaptor proteins. In this study, we investigated the interactions between RHOV and SH3 domain-containing adaptor proteins, including GRB2 and NCK2. The RHOV-induced serum response factor (SRF)-dependent gene transcriptional activity was attenuated in cells co-expressing either GRB2 or NCK2 compared to cells expressing RHOV alone. From the results of experiments using various gene mutants of RHOV and GRB2, it appears that the PR motif of the N-terminal region of RHOV is the crucial binding site for the SH3 domain-containing proteins. Furthermore, we found that Ser25 in the N-terminal region of RHOV is phosphorylated by PKA and that its phosphorylation is suppressed by interaction with NCK2 but not GRB2. We have found a novel regulatory mechanism for the phosphorylation of RHOV and its interaction with SH3 domain-containing adaptor proteins.

The Rho guanine nucleotide exchange factor PLEKHG1 is activated by interaction with and phosphorylation by Src family kinase member FYN.

Rho family small GTPases (Rho) regulate various cell motility processes by spatiotemporally controlling the actin cytoskeleton. Some Rho-specific guanine nucleotide exchange factors (RhoGEFs) are regulated via tyrosine phosphorylation by Src family tyrosine kinase (SFK). We also previously reported that PLEKHG2, a RhoGEF for the GTPases Rac1 and Cdc42, is tyrosine-phosphorylated by SRC. However, the details of the mechanisms by which SFK regulates RhoGEFs are not well understood. In this study, we found for the first time that PLEKHG1, which has very high homology to the Dbl and pleckstrin homology domains of PLEKHG2, activates Cdc42 following activation by FYN, a member of the SFK family. We also show that this activation of PLEKHG1 by FYN requires interaction between these two proteins and FYN-induced tyrosine phosphorylation of PLEKHG1. We also found that the region containing the Src homology 3 and Src homology 2 domains of FYN is required for this interaction. Finally, we demonstrated that tyrosine phosphorylation of Tyr-720 and Tyr-801 in PLEKHG1 is important for the activation of PLEKHG1. These results suggest that FYN is a regulator of PLEKHG1 and may regulate cell morphology through Rho signaling via the interaction with and tyrosine phosphorylation of PLEKHG1.

Histone modification dynamics as revealed by multicolor immunofluorescence-based single-cell analysis.

Post-translational modifications on histones can be stable epigenetic marks or transient signals that can occur in response to internal and external stimuli. Levels of histone modifications fluctuate during the cell cycle and vary among different cell types. Here, we describe a simple system to monitor the levels of multiple histone modifications in single cells by multicolor immunofluorescence using directly labeled modification-specific antibodies. We analyzed histone H3 and H4 modifications during the cell cycle. Levels of active marks, such as acetylation and H3K4 methylation, were increased during the S phase, in association with chromatin duplication. By contrast, levels of some repressive modifications gradually increased during G2 and the next G1 phases. We applied this method to validate the target modifications of various histone demethylases in cells using a transient overexpression system. In extracts of marine organisms, we also screened chemical compounds that affect histone modifications and identified psammaplin A, which was previously reported to inhibit histone deacetylases. Thus, the method presented here is a powerful and convenient tool for analyzing the changes in histone modifications.

H3K9me3 maintenance on a human artificial chromosome is required for segregation but not centromere epigenetic memory.

Most eukaryotic centromeres are located within heterochromatic regions. Paradoxically, heterochromatin can also antagonize de novo centromere formation, and some centromeres lack it altogether. In order to investigate the importance of heterochromatin at centromeres, we used epigenetic engineering of a synthetic alphoidtetO human artificial chromosome (HAC), to which chimeric proteins can be targeted. By tethering the JMJD2D demethylase (also known as KDM4D), we removed heterochromatin mark H3K9me3 (histone 3 lysine 9 trimethylation) specifically from the HAC centromere. This caused no short-term defects, but long-term tethering reduced HAC centromere protein levels and triggered HAC mis-segregation. However, centromeric CENP-A was maintained at a reduced level. Furthermore, HAC centromere function was compatible with an alternative low-H3K9me3, high-H3K27me3 chromatin signature, as long as residual levels of H3K9me3 remained. When JMJD2D was released from the HAC, H3K9me3 levels recovered over several days back to initial levels along with CENP-A and CENP-C centromere levels, and mitotic segregation fidelity. Our results suggest that a minimal level of heterochromatin is required to stabilize mitotic centromere function but not for maintaining centromere epigenetic memory, and that a homeostatic pathway maintains heterochromatin at centromeres.This article has an associated First Person interview with the first authors of the paper.

CENP-B creates alternative epigenetic chromatin states permissive for CENP-A or heterochromatin assembly.

CENP-B binds to CENP-B boxes on centromeric satellite DNAs (known as alphoid DNA in humans). CENP-B maintains kinetochore function through interactions with CENP-A nucleosomes and CENP-C. CENP-B binding to transfected alphoid DNA can induce de novo CENP-A assembly, functional centromere and kinetochore formation, and subsequent human artificial chromosome (HAC) formation. Furthermore, CENP-B also facilitates H3K9 (histone H3 lysine 9) trimethylation on alphoid DNA, mediated by Suv39h1, at ectopic alphoid DNA integration sites. Excessive heterochromatin invasion into centromere chromatin suppresses CENP-A assembly. It is unclear how CENP-B controls such different chromatin states. Here, we show that the CENP-B acidic domain recruits histone chaperones and many chromatin modifiers, including the H3K36 methylase ASH1L, as well as the heterochromatin components Suv39h1 and HP1 (HP1α, ß and γ, also known as CBX5, CBX1 and CBX3, respectively). ASH1L facilitates the formation of open chromatin competent for CENP-A assembly on alphoid DNA. These results indicate that CENP-B is a nexus for histone modifiers that alternatively promote or suppress CENP-A assembly by mutually exclusive mechanisms. Besides the DNA-binding domain, the CENP-B acidic domain also facilitates CENP-A assembly de novo on transfected alphoid DNA. CENP-B therefore balances CENP-A assembly and heterochromatin formation on satellite DNA.

Regulation of RNA polymerase II activation by histone acetylation in single living cells.

In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells. Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our single-cell analysis reveals histone H3 lysine-27 acetylation at a gene locus can alter downstream transcription kinetics by as much as 50%, affecting two temporally separate events. First acetylation enhances the search kinetics of transcriptional activators, and later the acetylation accelerates the transition of RNAP2 from initiation to elongation. Signatures of the latter can be found genome-wide using chromatin immunoprecipitation followed by sequencing. We argue that this regulation leads to a robust and potentially tunable transcriptional response.

DBS is activated by EPHB2/SRC signaling-mediated tyrosine phosphorylation in HEK293 cells.

It is well known that Rho family small GTPases (Rho GTPase) has a role of molecular switch in intracellular signal transduction. The switch cycle between GTP-bound and GDP-bound state of Rho GTPase regulates various cell responses such as gene transcription, cytoskeletal rearrangements, and vesicular trafficking. Rho GTPase-specific guanine nucleotide exchange factors (RhoGEFs) are regulated by various extracellular stimuli and activates Rho GTPase such as RhoA, Rac1, and Cdc42. The molecular mechanisms that regulate RhoGEFs are poorly understood. Our studies reveal that Dbl's big sister (DBS), a RhoGEF for Cdc42 and RhoA, is phosphorylated at least on tyrosine residues at 479, 660, 727, and 926 upon stimulation by SRC signaling and that the phosphorylation at Tyr-660 is particularly critical for the serum response factor (SRF)-dependent transcriptional activation of DBS by Ephrin type-B receptor 2 (EPHB2)/SRC signaling. In addition, our studies also reveal that the phosphorylation of Tyr-479 and Tyr-660 on DBS leads to the actin cytoskeletal reorganization by EPHB2/SRC signaling. These findings are thought to be useful for understanding pathological conditions related to DBS such as cancer and non-syndromic autism in future.

Four-and-a-half LIM Domains 1 (FHL1) Protein Interacts with the Rho Guanine Nucleotide Exchange Factor PLEKHG2/FLJ00018 and Regulates Cell Morphogenesis.

PLEKHG2/FLJ00018 is a Gßγ-dependent guanine nucleotide exchange factor for the small GTPases Rac and Cdc42 and has been shown to mediate the signaling pathways leading to actin cytoskeleton reorganization. Here we showed that the zinc finger domain-containing protein four-and-a-half LIM domains 1 (FHL1) acts as a novel interaction partner of PLEKHG2 by the yeast two-hybrid system. Among the isoforms of FHL1 (i.e. FHL1A, FHL1B, and FHL1C), FHL1A and FHL1B interacted with PLEKHG2. We found that there was an FHL1-binding region at amino acids 58-150 of PLEKHG2. The overexpression of FHL1A but not FHL1B enhanced the PLEKHG2-induced serum response element-dependent gene transcription. The co-expression of FHL1A and Gßγ synergistically enhanced the PLEKHG2-induced serum response element-dependent gene transcription. Increased transcription activity was decreased by FHL1A knock-out with the CRISPR/Cas9 system. Compared with PLEKHG2-expressing cells, the number and length of finger-like protrusions were increased in PLEKHG2-, Gßγ-, and FHL1A-expressing cells. Our results provide evidence that FHL1A interacts with PLEKHG2 and regulates cell morphological change through the activity of PLEKHG2.

CENP-C and CENP-I are key connecting factors for kinetochore and CENP-A assembly.

Although it is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity, the pathways leading to the formation and maintenance of centromere chromatin remain unclear. We previously generated human artificial chromosomes (HACs) whose centromeres contain a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator (alphoid(tetO)). We also obtained cell lines bearing the alphoid(tetO) array at ectopic integration sites on chromosomal arms. Here, we have examined the regulation of CENP-A assembly at centromeres as well as de novo assembly on the ectopic arrays by tethering tetracycline repressor (tetR) fusions of substantial centromeric factors and chromatin modifiers. This analysis revealed four classes of factors that influence CENP-A assembly. Interestingly, many kinetochore structural components induced de novo CENP-A assembly at the ectopic site. We showed that these components work by recruiting CENP-C and subsequently recruiting M18BP1. Furthermore, we found that CENP-I can also recruit M18BP1 and, as a consequence, enhances M18BP1 assembly on centromeres in the downstream of CENP-C. Thus, we suggest that CENP-C and CENP-I are key factors connecting kinetochore to CENP-A assembly.

Threonine 680 phosphorylation of FLJ00018/PLEKHG2, a Rho family-specific guanine nucleotide exchange factor, by epidermal growth factor receptor signaling regulates cell morphology of Neuro-2a cells.

FLJ00018/PLEKHG2 is a guanine nucleotide exchange factor for the small GTPases Rac and Cdc42 and has been shown to mediate the signaling pathways leading to actin cytoskeleton reorganization. The function of FLJ00018 is regulated by the interaction of heterotrimeric GTP-binding protein Gßγ subunits or cytosolic actin. However, the details underlying the molecular mechanisms of FLJ00018 activation have yet to be elucidated. In the present study we show that FLJ00018 is phosphorylated and activated by ß1-adrenergic receptor stimulation-induced EGF receptor (EGFR) transactivation in addition to Gßγ signaling. FLJ00018 is also phosphorylated and activated by direct EGFR stimulation. The phosphorylation of FLJ00018 by EGFR stimulation is mediated by the Ras/mitogen-activated protein kinase (MAPK) pathway. Through deletion and site-directed mutagenesis studies, we have identified Thr-680 as the major site of phosphorylation by EGFR stimulation. FLJ00018 T680A, in which the phosphorylation site is replaced by alanine, showed a limited response of the Neuro-2a cell morphology to EGF stimulation. Our results provide evidence that stimulation of the Ras/MAPK pathway by EGFR results in FLJ00018 phosphorylation at Thr-680, which in turn controls changes in cell shape.

Mastermind-like 1 (MamL1) and mastermind-like 3 (MamL3) are essential for Notch signaling in vivo.

Mastermind (Mam) is one of the elements of Notch signaling, a system that plays a pivotal role in metazoan development. Mam proteins form transcriptionally activating complexes with the intracellular domains of Notch, which are generated in response to the ligand-receptor interaction, and CSL DNA-binding proteins. In mammals, three structurally divergent Mam isoforms (MamL1, MamL2 and MamL3) have been identified. There have also been indications that Mam interacts functionally with various other transcription factors, including the p53 tumor suppressor, ß-catenin and NF-κB. We have demonstrated previously that disruption of MamL1 causes partial deficiency of Notch signaling in vivo. However, MamL1-deficient mice did not recapitulate total loss of Notch signaling, suggesting that other members could compensate for the loss or that Notch signaling could proceed in the absence of Mam in certain contexts. Here, we report the generation of lines of mice null for MamL3. Although MamL3-null mice showed no apparent abnormalities, mice null for both MamL1 and MamL3 died during the early organogenic period with classic pan-Notch defects. Furthermore, expression of the lunatic fringe gene, which is strictly controlled by Notch signaling in the posterior presomitic mesoderm, was undetectable in this tissue of the double-null embryos. Neither of the single-null embryos exhibited any of these phenotypes. These various roles of the three Mam proteins could be due to their differential physical characteristics and/or their spatiotemporal distributions. These results indicate that engagement of Mam is essential for Notch signaling, and that the three Mam isoforms have distinct roles in vivo.

WDR11, a WD protein that interacts with transcription factor EMX1, is mutated in idiopathic hypogonadotropic hypogonadism and Kallmann syndrome.

By defining the chromosomal breakpoint of a balanced t(10;12) translocation from a subject with Kallmann syndrome and scanning genes in its vicinity in unrelated hypogonadal subjects, we have identified WDR11 as a gene involved in human puberty. We found six patients with a total of five different heterozygous WDR11 missense mutations, including three alterations (A435T, R448Q, and H690Q) in WD domains important for ß propeller formation and protein-protein interaction. In addition, we discovered that WDR11 interacts with EMX1, a homeodomain transcription factor involved in the development of olfactory neurons, and that missense alterations reduce or abolish this interaction. Our findings suggest that impaired pubertal development in these patients results from a deficiency of productive WDR11 protein interaction.

The importance of interaction with membrane lipids through the pleckstrin homology domain of the guanine nucleotide exchange factor for rho family small guanosine triphosphatase, FLJ00018.

FLJ00018, a heterotrimeric guanosine 5'-triphosphate (GTP)-binding protein (G protein) Gßγ subunit-activated guanine nucleotide exchange factor for Rho family small GTPases, regulates cellular responses, including cell morphological changes and gene transcriptional regulation, and targets the cellular membranes. FLJ00018 contains a Dbl homology (DH) domain in addition to a pleckstrin homology (PH) domain. Here we show that the PH domain of FLJ00018 is required for FLJ00018-induced, serum response element-dependent gene transcription. Although the PH domain of KIAA1415/P-Rex1, another Gßγ subunit-activated guanine nucleotide exchange factor for Rho family small GTPases, binds to phosphatidylinositol 3,4,5-triphosphate and phosphatidylinositol 3,4-bisphosphate, the PH domain of FLJ00018 binds to polyphosphoinositides including phosphatidylinositol 4,5-bisphosphate, and phosphatidic acid. These results suggest that FLJ00018 is targeted via its PH domain to cellular membranes.

Characterization of the expression and cell-surface localization of transmembrane protein 132A.

Transmembrane protein 132A (TMEM132A, KIAA1583) was first isolated as a novel gene that is enriched during the embryonic and postnatal stages of rat brain development and interacts with GRP78. However, the biological functions of TMEM132A are scarcely characterized because the protein does not contain any known structural domains. Using a cell-surface biotinylation assay and immunocytochemical staining, we found that TMEM132A is a transmembrane glycoprotein consisting of a large extracellular domain in the N-terminal region and a small cytosolic domain in the C-terminal region. Partial deletions of the intracellular domain of TMEM132A had little effect on its expression level and cell-surface localization in transfected HEK293 cells, whereas deletions of the extracellular domain hampered transport to the cell surface. The expression pattern of each N-terminal mutant was immunocytochemically confirmed in HeLa cells transfected with the same constructs. Treatment with tunicamycin, an inhibitor of protein glycosylation, led to the accumulation of the unglycosylated form of TMEM132A in inverse proportion to the glycosylated form; however, both forms were localized at the cell surface at almost equal rates. In contrast, GRP78 overexpression led to the accumulation of unglycosylated TMEM132A, which was not detected on the cell surface. Inhibition of ER-Golgi transport by treatment with brefeldin A or the overexpression of mutant Sar1 attenuated the amount of cell-surface localized TMEM132A in HEK293 cells. Treatment with reagents disrupting intracellular calcium rapidly down-regulated the amount of TMEM132A protein in Neuro2a cells without affecting the expression level of its mRNA. Taken together, our data show that the novel cell-surface localized glycoprotein, TMEM132A, is regulated by several factors, including GRP78, Sar1, and intracellular calcium, in a post-transcriptional manner.

Gs and Gq signalings regulate hPEM-2-induced cell responses in Neuro-2a cells.

Rho family GTPase-specific guanine nucleotide exchange factors of the Dbl family regulate a variety of cellular events including cytoskeletal arrangement, signal transduction, and gene expression through activation of Rho family GTPases. In this study, we show that hPEM-2 is a downstream effector of G(s) and G(q) signaling in Neuro-2a neuroblastoma cells. Co-expression with hPEM-2 and GTPase-deficient (constitutively active) mutants of Gαs (Gα(s)Q213L) or Gα(q) (Gα(q)Q209L), but not other GTPase-deficient mutants of Gα subunit and Gßγ subunits, activated serum response element (SRE)-dependent gene transcription, which is known to be induced by Rho family activation. Although a dominant negative mutant of Rac1 strongly blocks Gα(s)Q213L or Gα(q)Q209L/hPEM-2 activated SRE-dependent gene transcription, those of Cdc42 or RhoA are marginally affected. A PKA inhibitor, H-89, attenuated Gα(s)/hPEM-2-activated SRE-dependent gene transcription. And a dominant negative mutant of c-Src and an Src inhibitor attenuated Gα(q)Q209L/hPEM-2-activated SRE-dependent gene transcription. Experiments using hPEM-2 deletion mutants indicate that some regions of hPEM-2 play an important role in enhancing SRE activation by G(s) and G(q) signalings. These results reveal that G(s) and G(q) signalings regulate hPEM-2 functions through PKA and c-Src in Neuro-2a neuroblastoma cells, respectively.

An Artificial Conversion of Roots into Organs with Shoot Stem Characteristics by Inducing Two Transcription Factors.

Somatic plant cells can regenerate shoots and/or roots or adventitious embryonic calluses, which may induce organ formation under certain conditions. Such regenerations occur via dedifferentiation of somatic cells, induction of organs, and their subsequent outgrowth. Despite recent advances in understanding of plant regeneration, many details of shoot induction remain unclear. Here, we artificially induced shoot stem-like green organs (SSOs) in Arabidopsis thaliana roots via simultaneous induction of two transcription factors (TFs), ARABIDOPSIS THALIANA HOMEOBOX PROTEIN 25 (ATHB25, At5g65410) and the B3 family transcription factor REPRODUCTIVE MERISTEM 7 (REM7, At3g18960). The SSOs exhibited negative gravitropism and differentiated vascular bundle phenotypes. The ATHB25/REM7 induced the expression of genes controlling shoot stem characteristics by ectopic expression in roots. Intriguingly, the restoration of root growth was seen in the consecutive and adjacent parts of the SSOs under gene induction conditions. Our findings thus provide insights into the development and regeneration of plant shoot stems.

KAP1-independent transcriptional repression of SCAN-KRAB-containing zinc finger proteins.

The Krüppel-associated box-containing zinc finger gene family (KRAB-ZNF) is one of the largest gene families of transcriptional factors in the human genome. Although the functions of most of these genes remain to be determined, it is known that KRAB-mediated transcriptional repression requires a direct interaction with the KAP1 co-repressor. By mammalian one- or two-hybrid experiments in HEK293 cells, we compared transcriptional repression activities of 61 human KRAB-ZNFs. The results showed that six SCAN-KRAB-containing ZNFs are KAP1-independent transcriptional repressors whose SCAN-KRAB domain is unable to associate with KAP1 despite retaining transcriptional repression activity. Transcriptional repression activities of the SCAN-KRAB domain of KAP1-independent KRAB-ZNFs are not influenced by depletion of endogenous KAP1 levels by small interfering RNA. Although the mechanism by which KAP1-independent KRAB-ZNFs repress transcriptional activity remains to be elucidated, it appears that there may be a pathway for transcriptional repression that does not involve KAP1. These results provide new insight into the functions of the members of the KRAB-ZNF family.

Specific activation of PLEKHG2-induced serum response element-dependent gene transcription by four-and-a-half LIM domains (FHL) 1, but not FHL2 or FHL3.

PLEKHG2 is a Gßγ- and Gαs-dependent guanine nucleotide exchange factor for Rac1 and Cdc42 small GTPases and has been shown to mediate signaling pathways such as those for actin cytoskeletal reorganization and serum response element (SRE)-dependent gene transcription. We have shown that the four-and-a-half LIM domains (FHL) 1 acts as a positive regulator of PLEKHG2. Here, we evaluated the other FHL family members and found that the FHL1A specifically regulate the PLEKHG2 activity. Moreover, FHL1A further enhanced Gßγ- and PLEKHG2-induced SRE-dependent gene transcription, whereas FHL1A partially restored the attenuated PLEKHG2-induced SRE-dependent gene transcription by Gαs. Our results suggest that FHL1A specifically interacts with PLEKHG2 to regulate a function of PLEKHG2 that is modified by the interaction of Gßγ and Gαs.

The interaction between PLEKHG2 and ABL1 suppresses cell growth via the NF-κB signaling pathway in HEK293 cells.

The Rho family small GTPases mediate cell responses through actin cytoskeletal rearrangement. We previously reported that PLEKHG2, a Rho-specific guanine nucleotide exchange factor, is regulated via interaction with several proteins. We found that PLEKHG2 interacted with non-receptor tyrosine kinase ABL1, but the cellular function remains unclear. Here, we show that the interaction between PLEKHG2 and ABL1 attenuated the PLEKHG2-induced serum response element-dependent gene transcription in a tyrosine phosphorylation-independent manner. PLEKHG2 and ABL1 were co-localized and accumulated within cells co-expressing PLEKHG2 and ABL1. The cellular fractionation analysis suggested that the accumulation involved actin cytoskeletal reorganization. We also revealed that the co-expression of PLEKHG2 with ABL1, but not BCR-ABL, suppressed cell growth and synergistically enhanced NF-κB-dependent gene transcription. The cell growth suppression was canceled by co-expression with IκBα, a member of the NF-κB inhibitor protein family. This study suggests that the interaction between PLEKHG2 and ABL1 suppresses cell growth through intracellular protein accumulation via the NF-κB signaling pathway.

Exploration of human ORFeome: high-throughput preparation of ORF clones and efficient characterization of their protein products.

In this study, we established new systematic protocols for the preparation of cDNA clones, conventionally termed open reading frame (ORF) clones, suitable for characterization of their gene products by adopting a restriction-enzyme-assisted cloning method using the Flexi cloning system. The system has following advantages: (1) preparation of ORF clones and their transfer into other vectors can be achieved efficiently and at lower cost; (2) the system provides a seamless connection to the versatile HaloTag labeling system, in which a single fusion tag can be used for various proteomic analyses; and (3) the resultant ORF clones show higher expression levels both in vitro and in vivo. With this system, we prepared ORF clones encoding 1,929 human genes and characterized the HaloTag-fusion proteins of its subset that are expressed in vitro or in mammalian cells. Results thus obtained have demonstrated that our Flexi ORF clones are efficient for the production of HaloTag-fusion proteins that can provide a new versatile set for a variety of functional analyses of human genes.